GIFT  OF 


SEMICENTENNIAL  PUBLICATIONS 

OF  THE 

UNIVERSITY  OF  CALIFORNIA 


1868-1918 


THE   PHYSICAL   CHEMISTET 

OF 

THE   PROTEINS 


"  From  the  point  of  view  of  the  physicist,  a  theory  of  matter  is  a 
policy  rather  than  a  creed;  its  object  is  to  connect  or  co-ordinate 
apparently  diverse  phenomena,  and  above  all  to  suggest,  stimu- 
late and  direct  experiment.  It  ought  to  furnish  a  compass  which, 
if  followed,  will  lead  the  observer  further  and  further  into  pre- 
viously unexplored  regions.  Whether  these  regions  will  be  barren 
or  fertile  experience  alone  will  decide;  but,  at  any  rate,  one  who 
is  guided  in  this  way  will  travel  onward  in  a  definite  direction,  and 
will  not  wander  aimlessly  to  and  fro."  J.  J.  Thomson,  "The 
Corpuscular  Theory  of  Matter," 


THE  PHYSICAL  CHEMISTRY 

OF 

THE  PROTEINS 


BY 


T.  BRAILSFORD  ROBERTSON,  PH.D.,  D.Sc. 

•f 

Professor  of  Biochemistry  and  Pharmacology  in 
the  University  of  California, 


LONGMANS,    GREEN   AND    CO. 

FOURTH  AVENUE  &  SOxn  STREET,  NEW  YORK 

39  PATERNOSTER  ROW,  LONDON 

BOMBAY,  CALCUTTA,  AND  MADRAS 

1918 


TU 


COPYRIGHT,  1918, 

BY 
LONGMANS,  GREEN  &  CO. 


DEDICATED  TO  MY  MOTHER 

IN   AFFECTIONATE  ACKNOWLEDGMENT 

OF  ENCOURAGEMENT  AND 

INSPIRATION 


420161 


PREFACE 


THE  Proteins,  with  which,  as  its  title  indicates,  this  work  has 
most  particularly  to  deal,  have,  ever  since  the  publication  of  the 
classic  researches  of  Graham,  been  generally  recognized  as  typical 
examples  of  that  class  of  substances  which  Graham  designated 
"Colloids."  This  work,  therefore,  although  primarily  concerned 
with  the  physical  chemistry  of  a  limited  section  of  the  class,  may 
also,  in  some  measure,  be  regarded  as  contributing  to  an  analysis 
of  the  properties  and  behavior  of  colloids  in  general,  in  so  far  as 
these  permit  of  illustration  by  the  properties  and  behavior  of 
the  various  members  of  the  protein  group. 

The  leading  problems  in  every  field  of  chemical  investigation  are, 
to  a  large  extent,  determined  by  the  salient  properties  of  the  sub- 
stances which  form  the  subject  of  inquiry,  and  for  this  reason  a 
thorough  acquaintance  with  what  may  be  termed  the  "  descriptive 
chemistry"  of  any  group  of  substances  is  prerequisite  to  a  suc- 
cessful attempt  to  interpret  their  behavior.  If  we  were  to  en- 
deavor to  interpret  the  behavior  of  the  proteins  solely  in  the  light 
of  preconceptions  derived  from  the  study  of  the  chemistry  of  the 
metals,  for  example,  or  the  simpler  organic  compounds,  we  would 
find  that  the  behavior  of  the  proteins  displays  merely  a  bewilder- 
ing variety  of  inconsistencies.  Their  "amphoteric"  character, 
their  multiple  basicity  and  acidity,  their  instability  in  aqueous  solu- 
tion and,  above  all,  the  enormous  mass  and  catenary  configuration 
of  their  molecules,  confer  upon  them  properties  which  are  com- 
paratively unfamiliar  in  other  fields  of  chemistry  or  else  exaggerate 
properties  which  are  normally  displayed  by  the  simpler  chemical 
bodies  only  to  a  comparatively  negligible  degree. 

The  proteins  in  this  respect  are  not  exceptional.  Thus  the  mode 
of  investigation  and  the  interpretation  of  the  behavior  of  the 
various  lipoids  must  primarily  be  conditioned  by  their  very  general 
insolubility  in  water  and  instability  in  the  presence  of  oxygen. 
The  behavior  of  the  complex  polysaccharides  is  conditioned  by 
their  relative  stability  and  by  the  extraordinary  tendency  to  op- 

vii 


viii  PREFACE 

tical  isomerism  which  is  displayed  by  the  simpler  carbohydrate 
radicals  out  of  which  they  are  built  up,  while,  on  the  other  hand, 
the  behavior  of  rubber  and  its  congeners  is  primarily  conditioned 
by  the  enormous  internal  molecular  friction  which  leads  them  to 
display,  to  an  exaggerated  extent,  phenomena  analogous  to  hys- 
teresis which,  although  not  wholly  negligible  in  other  colloids,  for 
example  in  the  protein  group,  nevertheless  seldom  present  them- 
selves as  salient  characteristics  of  their  behavior. 

The  colloids  are  therefore  an  exceedingly  heterogeneous  group, 
the  only  common  distinguishing  characteristic  being  the  relatively 
enormous  mass  and  volume  of  the  molecules  of  the  most  "typical" 
representatives  of  the  class,  and  of  course  as  many  gradations  of 
behavior  exist  as  there  are  gradations  in  the  mass  and  volume  of 
molecules.  Nothing  is  to  be  gained,  therefore,  by  endeavoring  to 
force  the  various  members  of  the  colloid  group  into  artificial  con- 
formity with  definitions  which  are  designed  to  separate  them,  as 
if  they  were  a  homogeneous  group,  from  other  classes  of  chemical 
substances.  To  describe  a  particular  property  or  mode  of  be- 
havior as  a  " colloidal  phenomenon"  neither  defines  nor  inter- 
prets it  and  furthermore  fails  even  to  describe  it,  since  there  are 
no  phenomena  which  are  distinctively  " colloidal"  and  displayed 
by  every  member  of  the  colloid  group,  saving  only  those  phenomena 
which  depend  primarily  upon  the  simple  factor  of  the  mass  or 
volume  of  the  molecules,  and  which  are  therefore  predictable 
from  and  implied  in  the  properties  or  behavior  of  the  smaller 
molecules  of  the  non-colloidal  substances. 

Similarly,  the  use  of  the  term  "adsorption"  to  describe  the 
union  between  colloids  and  other  substances  implies  a  uniformity 
where  no  uniformity  exists  and  is,  moreover,  devoid  of  utility  or 
meaning  unless  we  attach  to  the  definition  some  distinct  idea  of 
the  nature  of  the  underlying  forces  which  condition  the  union, 
whether  these  forces  be  regarded  as  consisting  of  chemical  (i.e.y 
atomic)  attractions  or  of  capillary  (i.e.,  molecular)  attractions. 
But  in  forming  such  concrete  ideas  we  are  simply  returning  to 
conceptions  which  are  already  familiar  to  us  in  the  "crystalloid" 
field  of  chemistry  and  which  call  for  no  definitions  which  we  do  not 
already  possess  as  the  result'  of  our  general  acquaintance  with  the 
physical  and  chemical  phenomena  which  are  displayed  by  simpler 
and  hitherto  more  familiar  substances. 

The  investigations  of  recent  years,  not  only  upon  the  behavior 


PREFACE  ix 

of  the  proteins,  but  upon  that  of  the  colloids  in  general,  have  re- 
sulted in  the  development  of  two  rather  sharply  differentiated 
schools  of  opinion.  The  one  school  endeavors,  so  far  as  technical 
difficulties  permit,  to  directly  apply,  with  modifications  suggested 
by  the  properties  and  structure  of  the  particular  colloid  under 
investigation,  the  known  laws  of  what  may  be  termed  "molec- 
ular" physical  chemistry  to  protein  and  other  colloidal  systems, 
while  the  other  school  hesitates  to  do  so.  The  essential  question 
at  issue  between  these  two  schools,  in  so  far  as  the  proteins  are 
concerned,  is,  I  think,  simply  this:  Are  we  justified  in  assuming 
that  the  rule  of  Avogadro  is  applicable  to  protein  solutions  or  are 
we  not?  In  other  words,  are  protein  solutions  molecularly  dis- 
persed systems  or  are  they,  rather,  suspensions  or  emulsions? 
The  latter  of  the  two  schools  to  which  I  have  referred  avers  from 
a  priori  considerations,  implicitly  or  explicitly,  that  Avogadro's 
rule  may  not  be  applied  to  protein  solutions,  or,  at  least,  that  its 
validity  for  these  solutions  should  be  established  before  we  venture 
to  apply  it.  The  former  school  prefers  to  assume  that  Avogadro's 
rule  does  apply  to  these  systems  until  actual  inapplicability 
demonstrates  that  it  does  not.  Now  the  "  proof  "  of  the  applica- 
bility of  Avogadro's  rule  to  systems  which  are  admittedly  molec- 
ularly dispersed  has  never  consisted  in  anything  but  the  appli- 
cability, to  these  systems,  of  laws  and  deductions  founded  upon 
Avogadro's  rule.  The  procedure  of  the  former  school  would 
appear,  therefore,  to  be  sound  and  well  justified  by  scientific 
precedent.  Following  this  procedure  we  will  be  enabled  to  cor- 
relate and  interpret  the  phenomena  which  are  exhibited  by  such 
protein  systems  as  may  chance  to  be  molecularly  dispersed,  while, 
on  the  other  hand,  we  shall  be  enabled  to  accurately  delimit  the 
conditions  under  which  molecularly  dispersed  protein  systems 
exist  and  those  under  which  they  do  not. 

In  this  work  I  have  endeavored  to  interpret  the  physico-chemi- 
cal behavior  of  the  proteins  in  the  light  of  the  laws  of  Boyle  and  of 
Gay-Lussac,  as  they  have  been  applied  to  solutions  by  van't  Hoff, 
and  of  the  Guldberg  and  Waage  mass-law  which,  as  Larmor  has 
shown,  is  a  direct  consequence  of  Avogadro's  rule  and  Boyle's 
law.*  I  have  also  assumed  the  validity,  in  protein  systems,  of 
the  first  and  second  laws  of  heat,  albeit  the  applicability  of  the 

*  Larmor,  J.,  Phil.  Trans.  Roy.  Soc.  London,  190  A  (1887),  p.  276.  T.  Brails- 
ford  Robertson,  Journ.  Physical  Chem.,  10  (1906),  p.  521. 


X  PREFACE 

second  law  of  heat  to  protein  systems  has,  in  some  quarters,*  been 
questioned.  In  considering  the  electrochemical  behavior  of  the 
proteins  I  have  assumed  the  applicability  of  Arrhenius'  hypothesis 
of  electrolytic  dissociation,  of  Kolrausch's  law  of  the  independent 
motion  of  ions,  of  the  Nernst  theory  of  concentration-cells,  and 
further,  although  this  has  of  recent  years  been  very  strongly 
questioned,!  the  applicability  of  the  Guldberg  and  Waage  mass- 
law  to  reactions  between  ions.  I  believe  that  the  utility  of  these 
hypotheses  justifies  us  in  applying  them  until  still  more  useful 
hypotheses  shall  have  been  elaborated  to  amplify  or  replace  them. 

A  previous  edition  of  this  work  appeared,  in  German,  six  years 
ago.J  Since  that  time  our  knowledge  of  the  physico-chemical 
behavior  of  the  proteins  has  very  considerably  expanded  and  in- 
creased in  exactitude.  Among  the  particularly  important  in- 
vestigations which  have  appeared  during  this  period  may  be  men- 
tioned the  invention  by  Van  Slyke  of  an  accurate  and  simple 
method  of  determining  free  amino-groups  (Cf.  Chapter  I),  the 
work  of  Pauli  upon  the  combining-capacity  of  deaminized  pro- 
teins (Cf.  Chapters  I,  VIII  and  IX),  the  work  of  Schmidt  and  of 
af  Ugglas  upon  compound  proteins  (Cf.  Chapter  VII),  the  work 
of  Procter  upon  the  swelling  of  protein  jellies  (Cf.  Chapter  XII), 
the  work  of  Reichert  and  Brown  upon  the  crystallography  of 
haemoglobin  (Cf.  Chapter  XII)  and  that  of  Dabrovsky  upon  the 
molecular  volumes  of  proteins  dissolved  in  water  and  in  solutions 
of  coagulating  salts  (Cf.  Chapter  XIII).  The  present  edition 
has  been  almost  entirely  rewritten  and  the  literature  has  been 
brought  down  to  the  middle  of  1917. 

In  conclusion  I  wish  to  express  my  very  great  indebtedness  to  my 
wife,  for  her  assistance  in  preparing  the  manuscript  for  the  press, 
to  Dr.  Hardolph  Wasteneys  for  his  assistance  in  proof-reading, 
and  to  Dr.  C.  L.  A.  Schmidt  for  his  assistance  in  proof-reading 
and  in  the  verification  of  many  references  and  formulas. 

T.  BRAILSFORD  ROBERTSON. 

BERKELEY,  CALIFORNIA, 
Nov.  1,  1917. 

*  von  Schroeder,  P.,  Zeit.  f.  physik.  Chem.,  45  (1903),  p.  75.  Dietz,  W., 
Zeit.  f.  physiol.  Chem.,  52  (1907),  p.  279.  Cf.,  however,  Chapter  XVII. 

t  Cf.  for  example,  W.  Sutherland,  Phil.  Mag.  Series  6,  14  (1907),  p.  1. 

t  "  Die  physikalische  Chemie  der  Proteine,"  Dresden,  1912.  Theodor  Stein- 
kopff. 


CONTENTS 

PAQB 

PREFACE vii 

PART  I.   CHEMICAL  STATICS  IN  PROTEIN  SYSTEMS 

CHAPTER  I.    THE  CHEMICAL  CONSTITUTION  OP  THE  PROTEINS 3 

1.  The  Chemical  Homogeneity  of  the  Protein  Group 3 

2.  The  Products  of  the  Decomposition  of  the  Proteins 4 

3.  The  Connection  between  the  Amino-acid  Content  and  the  Prop- 

erties of  the  Proteins 8 

4.  The  Synthesis  of  Proteins 9 

5.  The  Occurrence  of  Peptids  among  the  Products  of  Protein  Hy- 

drolysis   15 

6.  The  Analysis  and  Characterization  of  Proteins  by  the  Determi- 

nation of  the  Chemical  Groups  Characteristic  of  the  Different 

Amino-acids 16 

7.  Types  of  Union  in  the  Protein  Molecule 17 

8.  Some  Consequences  of  the  Polypeptid  Structure  of  the  Protein 

Molecule 20 

9.  "Racemized"  Proteins 30 

CHAPTER  II.     THE  PREPARATION  OF  PURE  PROTEINS 34 

1.  The  Proteins  as  Chemical  Individuals 34 

2.  Casein 37 

3.  "Insoluble"  Serum  Globulin 40 

4.  Fibrin 42 

5.  Haemoglobin .  • 43 

6.  Crystallizable  Egg  Albumin 44 

7.  Ovovitellin 44 

8.  The  Vegetable  Proteins 46 

9.  The  Alcohol  Soluble  Vegetable  Proteins  (Gliadin,  Zein,  etc.) 48 

10.  Ovomucoid 49 

11.  Gelatin  and  Deaminized  Gelatin 51 

12.  Globin 51 

13.  The  Protamins 53 

CHAPTER  III.    THE  QUANTITATIVE  ESTIMATION  OF  THE  PROTEINS..  .  56 

1.  The  General  Principles  Underlying  the  Quantitative  Estimation 

of  Proteins 56 

2.  The  Nephelometric  Method  of  Estimating  Proteins 58 

3.  The  Refractometric  Method  of  Estimating  Proteins 60 

xi 


xii  CONTENTS 

PAGE 

CHAPTER  IV.    THE  COMPOUNDS  OF  THE  PROTEINS 67 

1.  The  Amphoteric  Character  of  the  Proteins 67 

2.  The  Direct  Method  of  Demonstrating  the  Existence  of  Protein 

Compounds  by  Precipitation  or  Coagulation 68 

3.  The  Direct  Method  of  Demonstrating  the  Existence  of  Protein 

Compounds  by  the  Solution  of  otherwise  Insoluble  Substances.  71 

4.  The  Indirect  Method  of  Precipitation 71 

5.  The  Method  of  Electrical  Conductivity 74 

6.  The  Cryoscopic  Method 74 

7.  The  Potentiometric  Method 76 

8.  The  Method  of  Catalysis 79 

9.  The  Indicator  Method 81 

10.  The  Method  of  "Masking"  the  Physiological  Effects  of  Ions  by 

the  Addition  of  Proteins  to  their  Solutions 82 

CHAPTER  V.    THE  COMPOUNDS  OF  THE  PROTEINS  —  Continued 85 

1.  Stoichiometrical  Relations  in  Protein  Compounds 85 

2.  The  Compounds  of  the  Protamines  with  Inorganic  Acids  and 

Bases 86 

3.  The  Compounds  of  Casein  with  Bases,  Acids,  and  Salts 88 

4.  The  Compounds  of  Serum  Globulin  with  Inorganic  Acids  and 

Bases 98 

5.  The  Compounds  of  Fibrin  with  Inorganic  Acids  and  Bases 101 

6.  The  Compounds  of  the  Vegetable  Proteins  with  Inorganic  Acids 

and  Bases 101 

7.  The  Compounds  of  Ovomucoid  with  Acids  and  Bases 103 

8.  Summary  of  Some  of  the  Results  Cited  in  this  Chapter 103 

CHAPTER  VI.     THE  COMPOUNDS  OF  THE  PROTEINS  —  Continued 107 

1.  General  Remarks  on  the  Precipitation  of  the  Proteins  by  Inorganic 

Salts 107 

2.  Earlier  Investigations  on  the  Significance  of  the  State  of  Hydra- 

tion  of  the  Proteins  in  Relation  to  their  Coagulation  by  Salts.  108 

3.  The  Influence  of  the  Electrical  Condition  of  the  Proteins  upon 

their  Precipitation  and  Coagulation  by  Electrolytes 112 

4.  Later  Investigations  on  the  Significance  of  the  State  of  Hydration 

of  the  Proteins  in  Relation  to  their  Coagulation  by  Electro- 
lytes   119 

5.  Application  of  the  Phase-rule  to  Protein-salt-water  Systems 123 

6.  The  Chemical  Mechanics  of  the  Precipitation  and  Coagulation  of 

Proteins  by  Salts 125 

CHAPTER  VII.    THE  COMPOUNDS  OF  THE  PROTEINS  —  Continued 137 

1.  Compounds  with  the  Heavy  Metals 137 

2.  Compounds  with  the  Phosphoric  Acids 142 

3.  Compounds  of  the  Proteins  with  Carbonic  Acid 144 


CONTENTS  xiii 

PAGE 

4.  Compounds  of  the  Proteins  with  the  Alkaloidal  Reagents,  Dyes, 

Alkaloids,  etc 145 

5.  The  Compounds  of  Proteins  with  Soaps  and  Lipoids 148 

6.  The  Compounds  of  Proteins  with  Proteins  and  their  Possible 

Significance  in  Life-phenomena 148 

7.  Compounds  of  the  Proteins  with  Toxins,  Antibodies,  Ferments,  etc.  156 

8.  Methyl  and  Benzoyl  Derivatives  of  the  Proteins 156 

9.  The  Halogen-  and  Nitro-substitution  Compounds  of  the  Proteins.  157 

10.  The  Compounds  of  Proteins  with  Sulphur 159 

11.  The  Compounds  of  Proteins  with  Oxygen 159 


PART  II.    THE  ELECTROCHEMISTRY  OF  THE  PROTEINS 
CHAPTER  VIII.  THE  FORMATION  AND  DISSOCIATION  OF  PROTEIN  SALTS    167 

1.  Compounds  of  the  Proteins  with  Inorganic  Bases  and  Acids;  the 

Non-dissociable  Character  of  the  Inorganic  Radical 167 

2.  The  Electrolysis  of  Protein  Salts 176 

3.  The  Relative  Masses  of  Protein  Anions  and  Cations 184 

4.  The  Migration-velocity  of  Protein  Ions 187 

5.  Objections  to  the  above  Theory  of  Protein  lonization 189 

6»  Biological  Applications;  the  "Selective"  Action  of  Living  Tissues.  192 

CHAPTER  IX.    THE  COMBINING-CAPACITY  OF  THE  PROTEINS 195 

1.  The  Electrochemical  Determination  of  the  Combining-capacity 

of  the  Proteins 195 

2.  The  Combining-capacity  of  Casein  for  Bases  and  of  Ovomucoid 

for  HC1  at  Absolute  Neutrality 204 

3.  The  Non-dependence  of  the  Composition  of  the  Compounds  of 

Protein  with  Acids  and  Bases  upon  the  Dilution  of  their  Solu- 
tions      206 

4.  The  "Isoelectric"  Condition  of  Proteins  at  Certain  H+  and  OH' 

Concentrations 209 

5.  Biological  Applications;  the  Neutrality  of  the  Tissues  and  of  the 

Tissue-fluids 211 

CHAPTER  X.    THE  ELECTRICAL  CONDUCTIVITY  OF  SOLUTIONS  OF  PRO- 
TEIN SALTS 220 

1.  The  Influence  of  Dilution  upon  the  Conductivity  of  Solutions  of 

Protein  Salts 220 

2.  The  Depression  of  the  Freezing-point  of  Water  which  is  Caused  by 

Dissolved  Protein  Salts,  and  the  Stoichiometry  of  Protein  Salts.    236 

3.  The  Dependence  of  the  Electrical  Conductivity  of  Solutions  of 

Protein  Salts  upon  the  Proportion  of  Inorganic  Acid  or  Base 
which  the  Salts  Contain 242 

4.  The  Solubility  and  Minimal  Combining-capacities  of  Casein  and 

of  Serum  Globulin  in  Solutions  of  Bases. .  248 


Xiv  CONTENTS 

PAGE 
CHAPTER  XI.    THE  ELECTROCHEMISTRY  OF  COAGULATION 252 

1.  The  Coagulation  of  the  Casemates  by  Alcohol 252 

2.  The  Appli.cability.pf  the  Ostwald  Dilution-Law  to  Solutions  of  the 

Casemates  in  Alcohol-Water  Mixtures 254 

3.  The  Influence  of  the  Concentration  of  Alcohol  in  the  Solvent 

upon  the  Conductivities  of  Solutions  of  Potassium  Caseinate. .     257 

4.  The  Interpretation  of  the  Law  xv  =  ^ 262 

5.  The  Viscosities  of  Solutions  of  Potassium  Caseinate  in  Alcohol- 

Water  Mixtures 266 

6.  The  Molecular  Condition  of  Potassium  Caseinate  in  75%  Alcohol    268 

7.  The  Chemical  Mechanics  of  the  Coagulation  of  Proteins  by  Al- 

cohol. .  270 


PART  III.   THE  PHYSICAL  PROPERTIES  OF  PROTEIN  SYSTEMS 

CHAPTER  XII.    THE   PHENOMENA   WHICH   ACCOMPANY   CHANGES   IN 

THE  STATE  OF  AGGREGATION  OF  PROTEINS 275 

1.  The  Passage  of  Dry  Protein  into  Solution 275 

2.  The  Swelling  of  Protein  Jellies 292 

3.  The  Gelatinization  and  Coagulation  of  Proteins 299 

4.  The  Coagulation  of  Proteins  by  Heat,  Light  and  Hydrostatic 

Pressure 304 

5.  The  Crystallization  of  Proteins 311 

CHAPTER  XIII.    CERTAIN  PHYSICAL  PROPERTIES  OF  PROTEIN  SOLU- 
TIONS, ETC 320 

1.  The  Viscosity  of  Protein  Solutions 320 

2.  The  Cohesiveness  of  Protein  Solutions 328 

3.  The  Elasticity  of  Protein  Solutions 329 

4.  The  Diffusion  of  Proteins  in  Solution 330 

5.  The  Freezing-  and  Boiling-points  of  Protein  Solutions 331 

6.  The  Osmotic  Pressure  of  Proteins  in  Solution 336 

7.  The  Nature  of  Protein  Solutions 340 

8.  The  Opalescence  of  Protein  Solutions;  the  Tyndall  Effect 342 

9.  The  Surface  Tension  of  Protein  Solutions . . .  / 345 

10.  The  Formation  of  Surface-films  by  Dissolved  Proteins 345 

11.  The  Specific  Gravities  of  Protein  Solutions 348 

12.  The  Magnetic  Properties  of  the  Proteins 349 

13.  The  "Gold  Number"  of  Proteins 349 

CHAPTER  XIV.    OPTICAL  PROPERTIES  OF  PROTEIN  SOLUTIONS 355 

1.  The  Specific  Rotatory  Power  of  the  Proteins 355 

2.  The  Absorption  of  Light  by  Protein  Solutions 358 

3.  The  Refractive  Indices  of  Protein  Solutions 359 


CONTENTS  XV 

PART  IV.    CHEMICAL  DYNAMICS  IN  PROTEIN  SYSTEMS 

PAGE 

CHAPTER  XV.    THE  HYDROLYSIS  OF  THE  POLYPEPTIDS 371 

1.  The  Hydrolysis  of  Polypeptids  by  Proteolytic  Enzymes 371 

2.  The  Kinetics  of  the  Hydrolysis  of  Polypeptids  by  Proteolytic 

Enzymes 376 

3.  The  Order  in  which  Amino-acids  are  Split  off  from  Polypeptids  by 

Proteolytic  Ferments 385  - 

CHAPTER  XVI.    THE  HYDROLYSIS  OF  THE  PROTEINS 389 

1.  The  Proteolytic  Enzymes  as  Catalysors 4.389 

2.  The  Evidence  for  the  Existence  of  Intermediate  Compounds 

between  the  Proteolytic  Ferments  and  their  Substrates 398 

3.  The  Kinetics  of  Protein  Hydrolysis  by  Enzymes 400 

4.  The  Influence  of  Acid  and  Alkali  upon  the  Rate  of  Protein  Hy- 

drolysis by  Enzymes 410 

5.  The  Influence  of  Added  Substances  upon  the  Rate  of  Protein 

Hydrolysis  by  Enzymes 415 

6.  The  Action  of  the  Coagulating  Ferments,  Rennet  and  Thrombin.  416 

7.  The  Influence  of  Temperature  upon  the  Velocity  of  Protein  Hy- 

drolysis   419  — - 

CHAPTER  XVII.    THE  ENZYMATIC  SYNTHESIS  OF  PROTEINS 425  ^~ 

1.  The  Reversion  of  the  Hydrolysis  of  Proteins  by  Pepsin  and  Tryp- 

sin 425 

2.  The  Probable  Nature  of  the  Coaguloses  and  Plasteins 437 

3.  The  Chemical  Mechanics  of  the  Fermentative  Synthesis  of  Pro- 

teins   439 

4.  Reciprocal  Catalysis 444 

5.  The  Influence  of  Temperature  upon  the  Enzymatic  Synthesis 

of  Proteins 448 

6.  The  Thermodynamics  of  the  Enzymatic  Hydrolysis  and  Synthesis 

of  Proteins 450 

7.  Biological  Applications ./fTS^. 452 

[ 
APPENDIX.    THE  TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS 

IN  PROTEIN  SYSTEMS \ 456 

INDEX  OF  AUTHORS >x 469 

INDEX  OF  SUBJECTS 479 


PART  I 
CHEMICAL  STATICS  IN  PROTEIN  SYSTEMS 


CHAPTER  I 
THE  CHEMICAL  CONSTITUTION  OF  THE  PROTEINS 

1.  The  Chemical  Homogeneity  of  the  Protein  Group.  —  The 

chemical  similarity  between  the  properties  and  behavior  of  'the 
various  protein  bodies,  and  the  evidence  thus  afforded  of  their 
close  chemical  relationship,  early  led  protein  chemists  to  ascribe 
to  the  proteins  a  common  and  characteristic  chemical  structure, 
and  to  endeavor  to  anticipate  the  nature  of  this  structure. 

The  difficulty  of  securing  a  quantitative  separation  of  protein 
bodies  is  eloquent  of  their  chemical  similarity;  one  by  one  the 
standard  methods  of  separation  brought  forward  by  the  physio- 
logical chemist,  such  as  fractional  heat-coagulation,  fractional 
precipitation  by  salts,  etc.,  have  .been  shown  to  possess  but  a 
qualitative  value;  the  separation  is  seldom  or  never  complete, 
and  the  critical  temperatures,  concentrations,  etc.,  at  which  pre- 
cipitations occur  are  seldom  well-defined  points,  but  appear, 
rather,  to  represent  portions  of  a  continuous  curve  of  solubility. 
The  close  relationship  between  the  various  protein  bodies  is 
furthermore  evinced  by  the  similarity  of  their  physical  properties, 
and  by  the  almost  universal  applicability,  within  the  protein 
group,  of  such  typical  color  or  other  reactions  as  the  proteins 
display.  The  general  physico-chemical  characteristics  of  the  vari- 
ous proteins  resemble  one  another,  as  we  shall  see  in  subsequent 
chapters,  quite  as  closely  as  their  purely  physical  or  purely  chemi- 
cal characteristics.  One  of  the  most  striking  of  these  physico- 
chemical  characteristics  is  their  digestibility  by  the  proteolytic 
enzymes.  All  true  proteins  and  a  large  number  of  the  poly- 
amino-acids  are  hydrolysable  by  trypsin,  while  the  overwhelming 
majority  of  the  proteins  are  also  hydrolysable  by  pepsin.  The 
chemical  relationship  between  the  various  proteins  would  there- 
fore appear  to  be  even  more  intimate  than  that  between  the 
various  disaccharides,  since  the  enzymes  which  accelerate  the 
hydrolysis  of  these  are,  as  a  rule,  specific,  the  hydrolysis  of  each 

3 


^CHEMICAL  STATICS 


sugar  being  accelerated  to  a  high  degree  by  one  enzyme  and  by 
that  alone. 
2.  The  Products  of  the  Decomposition  of  the  Proteins.  —  The 

proteins  are  notoriously  liable  to  decomposition,  and  a  complete 
breaking  down  of  the  sparingly  diffusible,  difficultly  crystallizable 
proteins,  into  substances  which  readily  diffuse  and  crystallize  can 
be  brought  about  by  a  variety  of  agencies,  such  as  the  following: 

I.  Fusion  with  alkali. 

II.  Oxidation  with  permanganate,  chromic  acid,  etc. 

III.  The  action  of  halogens. 

IV.  Hydrolysis,  through  one  or  more  of  the  following  agencies: 

a.  Heating  in  acid  solution, 

6.  Heating  in  alkaline  solution, 

c.  Treatment  with  superheated  steam. 

d.  Treatment  with  enzymes. 

Of  all  these  methods  that  of  hydrolysis  is  the  most  satisfactory 
and  yields  the  most  uniform  and  readily  inter pretable  results. 
It  appears  that  whatever  be  the  method  of  hydrolysis  employed, 
the  end-result,  provided  the  hydrolysis  has  been  complete,  is 
the  same,  namely,  the  production  of  a  mixture  of  amino-acids. 

Incomplete  hydrolysis,  however,  results  in  the  production  of  a 
number  of  intermediate  substances,  variously  designated,  in  the 
order  of  decreasing  complexity,  proteoses  (albumoses),  peptones 
and  polypeptids.  The  hydrolysis  of  the  proteins,  therefore, 
occurs  in  stages,  just  as,  in  the  hydrolysis  of  starch,  intermediary 
stages  (the  dextrins  and  maltose)  are  passed  through,  before  the 
attainment  of  the  last  stage  of  hydrolysis  and  the  complete  con- 
version of  the  starch  into  glucose. 

It  was  early  recognized  that  a  predominating  proportion  of  the 
products  of  the  complete  hydrolysis  of  proteins  consists  of  amino- 
acids,  and  the  most  readily  detected  amino-acids,  leucin  and  tyro- 
sin,  were  discovered,  respectively  by  Proust  in  1818  (54),  andjby 
Liebig  in  1846  (44).  The  chemical  constitution  of  these  sub- 
stances, however,  was  a  discovery  of  much  later  date,  that  of 
leucin  having  been  established  in  1868  by  Hiifner  (31)  and  of 
tyrosin  in  1869  by  Earth  (3). 

The  older  methods  of  isolating  individual  amino-acids  from  the 
mixture  which  the  complete  hydrolysis  of %  a  protein  yields,  de- 


PRODUCTS  OF  DECOMPOSITION  5 

pended  upon  the  fractional  crystallization  either  of  the  free 
ammo-acids  or  of  their  salts.  They  were  not  even  approximately 
quantitative  and  the  isolation  and  identification  of  a  given  amino- 
acid  could  only  be  effected  with  certainty  when  that  acid  was 
present  in  relatively  large  amounts.  Until  1890  only  mono- 
amino-acids  were  known,  with  certainty,  to  occur  among  the 
products  of  protein  hydrolysis.  Then  Drechsel  (13)  discovered 
lysin  and  lysatinin,  Hedin  (29)  isolated  arginin  and  Kossel  (34) 
isolated  histidin  from  among  the  dissociation  products  of  proteins. 

The  attainment  of  our  present  relatively  extensive  knowledge 
of  the  nature  and  yield  of  the  products  of  the  hydrolysis  of  pro- 
teins is  an  achievement  of  the  past  twenty  years,  and  we  owe  it 
in  the  first  place  to  the  labors  of  Emil  Fischer  and  of  Kossel  and 
their  pupils. 

In  1900  Kossel  and  Kutscher  (39)  (40)  succeeded  in  working 
out  a  method  for  the  quantitative  separation  and  estimation  of 
the  diamino-acids  lysin,  arginin  and  histidin  which  Kossel  (35) 
calls  the  hexone  bases.*  The  method  depends  in  principle  upon 
the  precipitation  of  arginin  and  histidin  in  the  form  of  their 
silver  salts,  and  of  lysin  first  by  phosphotungstic  acid  and  then. 
by  picric  acid.  A  partial  but  not  quantitative  separation  of  the 
diamino-acids  from  the  monoamino-acids  in  a  protein  digest, 
can  be  procured  by  precipitation  of  the  former  with  phospho- 
tungstic acid. 

In  1901  Emil  Fischer  (15)  introduced  a  new  method  of  sepa- 
rating and  estimating  the  monoamino-acids,  which,  with  modi- 
fications suggested  by  experience,  is  the  one  to  which  we  owe  the 
greater  part  of  our  present  knowledge  of  these  products  of  protein 
hydrolysis.  The  hydrolysis  is  carried  out,  as  a  rule,  by  boiling 
with  hydrochloric  acid.f  The  amino-acids  which  result  are  then 
converted  into  their  esters  by  dissolving  them  in  alcohol  and 
esterifying  by  saturation  of  the  solution  with  dry  hydrochloric 
acid  gas.  The  mixed  esters  thus  obtained  are  separated  by 

*  Owing  to  the  fact  that  they  each  contain  six  atoms  of  carbon.  An 
especial  interest  attaches  to  these  substances,  since,  unlike  the  monoamino 
acids,  they  are  predominantly  basic  in  character,  and  their  relation  to  the 
hexoses  suggests  their  possible  importance  in  carbohydrate  metabolism. 

t  N.  Zelinsky  (74)  has  suggested  the  employment  of  formic  acid  as  a  hy- 
drolysing  agent.  According  to  this  investigator  the  hydrolysis  by  formic  acid 
is  much  more  rapid  than  that  which  is  brought  about  by  boiling  the  proteins 
with  hydrochloric  acid. 


6  CHEMICAL  STATICS 

fractional  distillation  in  vacuo*  The  different  fractions  which 
are  thus  obtained  each  contain  the  esters  of  only  a  few  amino- 
acids.  The  esters  in  each  fraction  are  now  reconverted  into  the 
free  amino-acids,  and  the  individual  acids  are  separated,  identi- 
fied and  estimated  by  appropriate  methods  which  differ  some- 
what for  the  different  acids. 

The  method,  except  so  far  as  glutamic  acid  is  concerned,  is 
by  no  means  quantitative.  The  results  which  are  obtained  are 
minimal  yields.  The  extremely  insoluble  amino-acids,  tyrosin, 
cystin,  and  diaminotrioxydodecanic  acid  are  separated  from  the 
digest  before  esterification,  and,  so  far  as  tyrosin  is  concerned, 
our  estimate  is  tolerably  quantitative. 

By  a  combination  of  these  methods  it  has  been  shown  that  the 
protein  molecule  is  built  up  of  a  series  of  amino-acids,  and  the 
following  amino-acids  have  been  isolated  from  amongst  the 
products  of  the  hydrolysis  of  various  proteins.f 

A.   Monoaminomonocarboxylic  Acids 

1.  Glycin:  C2H5NO2,  or  amino-acetic  acid: 

CH2.NH2.COOH. 

2.  Alanin:  C3HyNO2,  or  a-aminopropionic  acid: 

CH3.CH(NH2).COOH. 

3.  Valin:  C6HnNO2,  or  a-aminoisovalerianic  acid  • 

8  X  CH.CH(NH2)COOH. 

4.  Leucin:  C6Hi3NO2,  or  a-aminoisocaproic  acid: 

CH.CH2.CH(NH2)COOH. 


CH3X 


5.   Isoleucin:  C6Hi3NO2,  or  a-amino-/3-methyl-j8-ethylpropionic  acid: 
CH3, 

XCH.CH(NH2).COOH. 


6.  Phenylalanin  :  C9HnNO2,  or  /8-phenyl-a-aminopropionic  acid: 

C6H5.CH2.CH(NH2).COOH. 

7.  Tyrosin:     C9HUNO3,  or  0-parahydroxyphenyl-a-aminopropionic  acid: 

HO.C6H4.CH2.CH(NH2)COOH. 

*    N.  Zelinsky,  A.  Annenkov,  and  J.  Kulikov  (75)  accomplish  the  separation 
of  the  individual  amino-acids  by  first  neutralizing  the  excess  of  hydrochloric 
acid  with  lead  hydroxide  and  then  subjecting  the  mixed  hydrochlorides  of  the 
ethyl  esters  to  fractional  distillation. 
t  Cited  from  Aders  Plimmer  (53). 


PRODUCTS  OF  DECOMPOSITION 

8.  Serin:  C3H7NO3,  or  /S-hydroxy-a-aminopropionic  acid: 

CH2(OH).CH(NH2)COOH. 

9.  Cystin:   C6Hi2N2O4S2  or  dicystein,  or  di-(/3-thio-a-aminopropionic 

acid)  : 
HOOC.CH(NH2).CH2.S  -  S.CH2.CH(NH2).COOH 

B.   Monoaminodicarboxylic  Acids 

10.   Aspartic  acid,  C4H7NO4,  or  aminosuccinic  acid. 
HOOC.CH2.CH(NH2).COOH. 


11.  Glutamic  acid,  CsHgNO^  or  a-aminoglutaric  acid: 

HOOC.CH2.CH2.CH(NH2)COOH. 

C.   Diaminomonocarboxylic  Acids 

12.  Arginin,  C6Hi4N4O2,  or  a-amino-5-guanidinvalerianic  acid: 

/NH2 
HN  =  C( 

x  NH.CH2.CH2.CH2.CH(NH2)COOH. 

13.  Lysin:  CeHi4N2O2  or  a-e-diaminocaproic  acid: 

H2N.CH2.CH2.CH2.CH2.CH(NH2).COOH. 

D.    Diamino-oxy-monocarboxylic  Acids 

14.  Caseinic  acid,  or  diaminotrioxydodecanic  acid: 

C12H26N205. 

E.   Heterocyclic  Compounds 

15.  Histidin,  C6H9N3O2,  or  /3-iminazolyl-a-aminopropionic  acid: 

CH 

#  \ 
N        NH 

I          I 
CH  =  C.CH2.CH(NH2).COOH 

16.  Prolin,  C5H9NO2,  or  a-pyrrolidin  carboxylic  acid: 


I          I 

CH2  CH.COOH 
VH 

17.  Oxyprolin,  or  oxypyrrolidin  carboxylic  acid: 

C6H9N03. 

18.  Tryptophane,  CnHi2N2O2,  or  /3-indole-a-aminopropionic  acid: 

C.CH2.CH.NH2.COOH 

/  ^ 

C6H4    CH 


VH 


8  CHEMICAL  STATICS 

3.  The  Connection  between  the  Amino-Acid  Content  and  the 
Properties  of  the  Proteins.  —  The  various  proteins  are  found  to 
be  built  up  of  the  several  units  enumerated  above  together, 
possibly,  with  units  which  we  have  not  yet  succeeded  in  isolating. 
Some  of  the  proteins  are  built  up  out  of  all  of  these  units  in  vary- 
ing proportions,  others  out  of  a  lesser  number;  so  that  in  some 
cases  amino-acids  which  are  predominant  among  the  products 
of  the  hydrolysis  of  one  protein  are 'absent  from  among  the  prod- 
ucts of  the  hydrolysis  of  another.  Nevertheless  the  general 
relationship  of  the  various  units  and  their  occurrence  in  dif- 
ferent proportions  in  the  various  proteins  accounts  not  only 
for  the  differences,  but  also  for  the  similarity  between 
them. 

In  many  instances  a  definite  parallelism  can  be  traced  between 
the  chemical  and  physical  behavior  of  the  proteins  and  their 
amino-acid  content.  Thus  the  albumins,  which  are  soluble  in 
distilled  water  and  are  not  coagulated  by  half-saturation  of  their 
solutions  with  ammonium  sulphate,  contain  no  glycin,  while  the 
globulins  which  are  (when  uncombined  with  bases  or  acids) 
insoluble  in  distilled  water,  and  are  coagulated  by  half-saturation 
of  their  solutions  with  ammonium  sulphate,  do  contain  this 
amino-acid.  The  alcohol-soluble  vegetable  proteins  contain  only 
a  trace  (probably  attributable  to  associated  impurities)  of  glycin 
and  some  of  them" contain  no  lysin,  their  content  of  diamino-acids 
is  very  small,  while  their  content  of  glutamic  acid  and  of  prolin 
is  very  high.  The  phosphoproteins  (casein,  vitellin)  are  also 
rather  high  in  glutamic  acid.  Gelatin  is  characterized  by  its 
high  glycin  content  and  keratin  (the  protein  of  hair  and  feathers) 
by  its  high  cystin  content.  The  histones,  which  are  predomi- 
nantly basic  substances,  contain  about  30  per  cent  of  diamino- 
acids,  while  the  protamins,  which  are  still  more  predominantly 
basic,  contain  only  small  amounts  of  monoamino-acids,  sal- 
min  containing  over  80  per  cent  of  arginin,  while  sturin  con- 
tains 67  per  cent  of  its  nitrogen  as  arginin,  10  per  cent  in 
the  form  of  histidin  and  from  6  to  7  per  cent  in  the  form  of 
lysin  (41). 

Kossel  (35)  has  expressed  the  view  that  all  proteins  are  built 
up  around  a  protamin  nucleus  and  that  this  accounts  for  the 
fact  that  the  majority  of  the  proteins  yield,  on  hydrolysis,  a 
certain  proportion  of  diamino-acids. 


SYNTHESIS  OF  PROTEINS  9 

4.  The  Synthesis  of  Proteins.  —  The  marked  predominance 
of  amino-acids  in  the  products  of  protein  hydrolysis,  long  ago 
led  protein  chemists  to  surmise  that  the  amino-acid  structure, 
or  some  derivative  of  that  structure,  must  be  represented  in  a 
high  degree  in  the  protein  molecule,  and  it  was  in  following 
this  clue  that  Schiitzenberger  (64)  carried  out  one  of  the  earliest 
and  most  successful  attempts  to  synthesize  bodies  of  a  protein 
character.  Recognizing  that  the  decomposition  of  proteins  into 
amino-acids  is  essentially  a  phenomenon  of  hydrolysis,  he  re- 
garded dehydration  as  an  essential  feature  of  any  attempt  at 
protein  synthesis,  while  the  abundance  of  amino-acids  among 
the  products  of  protein  hydrolysis  and  the  presence  therein  of 
bodies  related  to  urea,  led  him  to  believe  that  protein  synthesis 
must  consist  in  the  linkage  of  amino-acids  with  molecules  of  urea 
and  the  elimination  of  water.  Accordingly  amino-acids  were 
mixed  with  urea  and  phosphorus  pentoxide  and  heated  to  125°  C. 
The  product  was  a  pasty  solid,  soluble  in  water  and  readily  pre- 
cipitated by  alcohol.  It  was,  furthermore,  precipitated  from  aque- 
ous solution  by  the  usual  protein  precipitants  and  gave  the  biuret 
and  xanthoproteic  reactions. 

This  experiment  of  Schiitzenberger's  left  us,  however,  very 
much  where  we  were,  so  far  as  real  knowledge  of  the  structure 
of  the  protein  molecule  is  concerned.  The  knowledge  of  the  fact 
that  a  mixture  of  amino-acids  and  urea  yields,  under  certain 
treatment,  a  body  or  bodies  more  or  less  closely  resembling  the 
proteins,  furnished  us  little  or  no  information  regarding  the 
structure  of  the  protein  molecule  which  we  did  not  already 
possess  in  the  fact  that  the  disintegration  products  of  the  pro- 
teins are  predominantly  amino-acids.  Prior  to  Schiitzenberger, 
Grimaux  (28)  had  shown  that  condensation-products  of  amino- 
benzoic  acid  and  of  aspartic  acid  (probably  comparable  with  the 
octaspartic  acid  of  Schiff  (62))  resemble  the  proteins  in  many 
of  their  properties;  but  these  experiments  also  threw  no  light 
upon  the  structure  of  the  protein  molecule  beyond  emphasizing 
the  already  sufficiently  evident  probability  that  the  amino-acid 
grouping  plays  an  important  part  in  the  building  up  of  the  protein 
molecule. 

The  clue  which  led,  through  a  series  of  remarkable  researches 
to  our  present  comparatively  extensive  knowledge  of  the  groupings 
within  the  protein  molecule,  was  obtained  by  Curtius  (9)  who, 


10  CHEMICAL  STATICS 

in  1883,   observed  that  ethyl  glycocollate,  in  watery  solution, 
tended  to  form  glycocoll  anhydride: 

(In  the  absence  of  water) 
NH2.CH2.COOH  +  C2H5OH  =  NH2.CH2.COOC2H5  +  H20. 

(Glycocoll)  (Ethyl  glycocollate) 

(In  the  presence  of  water) 
NH2.CH2.COOC2H5  +  NH2.CH2COOC2H5  = 

(Ethyl  glycocollate)  (Ethyl  glycocollate) 

/CH2-NH. 

O  =  C,  XC  =  0  +  2C2H5OH. 

NNH.CH2    / 

(Glycocoll  anhydride) 

Obviously,  if  the  closed  ring  representing  the  glycocoll  anhy- 
dride molecule  could  be  opened  up  without  destroying  the  sta- 
bility of  the  molecule,  a  new  amino-acid  would  be  formed,  one 
degree  more  complex  than  the  original  amino-acid  (glycocoll). 
This  possibility  occurred  to  Emil  Fischer,  and  he  found,  in  fact, 
that  if  the  glycocoll  anhydride  which  is  thus  prepared  be  boiled 
for  a  short  time  with  concentrated  hydrochloric  acid,  the  fol- 
lowing change  occurs  : 

,  CH2.NH  x  „  CH2.NH2.HC1 


NH.CH2  NH.CH2.COOH 

(Glycocoll  anhydride)  (Glycyl-glycin  chloride) 

On  now  treating  the  glycyl-glycin  chloride  with  silver  oxide, 
silver  chloride  is  precipitated  and  free  glycyl-glycin  is  obtained. 
(23).  If,  however,  the  glycocoll  anhydride  be  originally  treated 
with  alcoholic  instead  of  with  watery  HC1,  the  ethyl  ester  of 
glycyl-glycin  is  obtained: 

/CH2.NH,  .CH2NH2 


NH.CH2  NH.CH2.COOC2H5 

(Glycocoll  anhydride)  (Glycyl-glycin  ester) 

It  would  almost  appear,  therefore,  as  if  we  had  only  to  repeat 
this  cycle  of  operations  indefinitely  in  order  to  secure  the  most 
complex  poly-amino-acids;  but  this  is  not  so  easy  as  it  might 
appear  at  first  sight;  the  instability  of  amino-acids  consequent 
upon  the  high  reactivity  of  the  NH2  group,  and  the  consequent 
difficulty  of  obtaining  simple  anhydrides  renders  this  procedure 


SYNTHESIS  OF  PROTEINS  11 

impossible.  Moreover  the  anhydride-ring  is  in  many  cases  (e.g., 
leucin  anhydride)  very  difficult  to  break  up  when  it  has  once 
been  formed. 

In  the  search  for  methods  of  overcoming  these  difficulties, 
Fischer  found  that  the  instability  of  the  amino-acids  could  be 
eliminated  by  the  introduction  of  radicals  into  the  NH2  group, 
and  he  and  Fourneau  synthesized  phenyl-cyanate-glycyl-glycin 
and  carboxyethyl-glycyl-glycin  ester  (C2H5O.OC.NH.CH2.CO.- 
NH.CH2.CO.OC2H5)  which  are  both  chemically  stable*  bodies. 
In  subsequent  investigations  Fischer  found  that  by  gentle  heat- 
ing, combination  between  the  esters  of  the  carboxyethyl  amino- 
acids  and  other  ammo-acid  esters  could  be  directly  brought  about 
(25);  in  this  way  carboxyethyl-diglycyl-leucin  ester  was  formed 
(C2H5O.OC.NH.CH2CO.NH.CH2.CO.NH.CH.C4H9.CO.OC2H5). 

The  difficulty  was  here  encountered,  however,  that  the  car- 
boxyethyl group,  once  introduced,  cannot  be  eliminated  again. 

The  method  which  Fischer  devised  to  overcome  this  difficulty 
(25)  was  extremely  ingenious.  The  introduction  of  a  radical  into 
the  NH2  group  appeared  to  be  a  necessity,  forced  upon  us  by 
the  impossibility  of  otherwise  securing  simple  anhydrides  of  the 
acids.  It  occurred  to  Fischer,  however,  that  the  radical  thus 
introduced  into  the  NH2  group  might  itself  be  made  a  carrier  of 
amino-acid  groups  into  the  molecule.  This  anticipation  proved 
to  be  correct.  The  radical  which  Fischer  first  utilized  was  the 
chloracetyl  group  (C1CH2.CO— );  when  chloracetyl  is  allowed  to 
act  upon  glycyl-glycin  ester  (obtained  by  the  methods  described 
above)  chloracetyl-glycyl-glycin  ester  is  obtained : 

C1.CH2.COC1  +  NH2.CH2.CO.NH.CH2.COOC2H5 

(Chloracetyl  chloride)  (Glycyl-glycin  ester) 

=  C1CH2CO.NH.CH2.CO.NH.CH2.COOC2H5  +  HC1. 

(Chloracetyl  glycyl-glycin  ester) 

By  saponification  of  this  ester,  free  chloracetyl-glycyl-glycin  is 
obtained;  on  now  treating  this  with  a  concentrated  aqueous 
solution  of  ammonia,  the  Cl  atom  in  the  chloracetyl  group  be- 
comes replaced  by  an  —  NH2  group  and  diglycyl-glycin  is  obtained : 

C1CH2.CO.NH.CH2.CO.NH.CH2.COOC2H5  +  2NH3 

(Chloracetyl-glycyl-glycin  ester) 

=  NH2.CH2.CO.NH.CH2CO.NH.CH2.COOC2H5  +  NH4C1. 

(Diglycyl-glycin  ester) 


12  CHEMICAL  STATICS 

In  other  words,  the  chloracetyl  group,  introduced  to  protect 
the  NH2  group  of  the  amino-acid  is,  after  it  has  performed  its 
protective  function,  itself  transformed  into  an  amino-acid  group, 
through  the  replacement  of  the  halogen  atom  by  NH2.  Obvi- 
ously, other  halogen-containing  acid  groups  may  be  used  in  place 
of  chloracetyl,  and  in  this  way  a  great  variety  of  amino-acid  groups 
can  be  introduced  into  the  NH2  group.  Thus  Fischer  employs: 

Chloracetyl-chloride  for  the  introduction  of  glycyl. 
a-Bromopropionyl-chloride  for  the  introduction  of  alanyl. 
1-a-Bromopropionyl-chloride  for  the  introduction  of  d-alanyl. 
ex-Bromobutyryl-chloride  for  the  introduction  of  a-aminobutyryl. 
a-Bromisocapronyl-chloride  for  the  introduction  of  leucyl. 
a-Bromophenylacetyl-chloride  for  the  introduction  of  phenylglycyl. 
a-Bromohydrocinnamyl-chloride  for  the  introduction  of  phenylalanyl. 
a-Phenylbromopropionyl-chloride  for  the  introduction  of  phenylalanyl. 
a-5-Dibromovaleryl-chloride  for  the  introduction  of  prolyl. 

Fumaryl-chloride  for  the  introduction  of  asparagyl. 

By  this  method  the  chain  of  amino-acids  is  lengthened  at  the 
amino-group  end.  Theoretically,  it  appeared  possible  tcValso. 
lengthen  the  chain  at  the  carboxyl  end  of  the  molecule,  by  acting 
upon  the  esters  of  the  amino-acids  with  the  acid  chlorides  of 
other  amino-acids.  Until  1904,  however,  the  acid  chlorides  of 
amino-acids  were  unknown  and  all  attempts  to  prepare  them  had 
failed,  owing  to  the  same  reason  which  limits  the  use  of  the  first 
method  of  synthesizing  poly-amino-acids,  described  above,  namely 
the  reactivity  of  the  NH2  group.  It  will  be  recollected  that 
Fischer  found  that  the  NH2  group  could  be  protected  by  the 
introduction  of  radicals,  and,  utilizing  this  fact,  in  1904  Fischer 
succeeded  in  devising  a  method  of  preparing  the  acid  chlorides 
of  the  amino-acids  (17).  The  acid  chlorides  thus  prepared  react 
with  the  esters  of  other  amino-  or  poly-amino-acids  to  form  poly- 
ami  no-acid  chains  of  greater  length.  Thus: 

C4H9.CHBr.CO.NH.CH2COCl  +  2  NH2.CH2.COOC2H5 

(Bromisocapronylglycyl  chloride)  (Glycin  ester) 

=  HC1NH2.CH2.COOC2H5  +  C4H9.CHBr.CO.NH.CH2CO.- 

NH.CH2COOC2H5. 

(Glycin  ester  hydrochloride)  (Bromisocapronylglycyl-glycin  ester) 

Subsequent  saponification  of  the  bromisocapronyl-glycyl-glycin 
ester  and  treatment  with  ammonia  yields  the  poly-amino-acid  (tri- 
peptid)  leucyl-glycyl-glycin: 

C4H9.CH(NH2).CO.NH.CH2.CO.NH.CH2.COOH. 


SYNTHESIS  OF  PROTEINS  13 

If  the  bromisocapronylglycylchloride  be  made  to  act  upon  glycyl- 
glycin  ester,  and  the  product  be  treated  in  the  same  way,  the 
tetrapeptid,  leucyl-diglycyl  glycin  results: 

C4H9.CH(NH2).CO.NH.CH2CO.NH.CH2CO.NH.CH2.COOH. 

These  methods  of  synthesis  proved  inadequate  where  hydroxy- 
amino-acids  are  concerned,  because  the  phosphorus  penta- 
chloride,  used  in  the  formation  of  the  acid  chloride,  attacks  the 
OH  group.  It  was,  however,  ascertained  that  the  OH  could  be 
protected  by  acting  upon  the  hydroxy  amino-acid  with  methyl 
chlorcarbonate  (20),  which  converts  the  OH  into  —  OC02CH3, 
which  is  not  attacked  by  PCU  and  is  readily  removed  by  hy- 
drolysis. In  this  way  it  has  proved  possible  to  introduce  phenyl- 
carboxylic  acids,  such  as  tyrosin,  into  poly-amino-acid  chains. 

By  these  methods,  and  modifications  of  these  methods,  Fischer 
has  succeeded  in  building  up  long  chains  of  amino-acid  groups, 
these  chains  being  collectively  termed,  by  Fischer,  peptids. 
Chains  consisting  of  two  links,  i.e.,  combinations  of  two  amino- 
acids,  Fischer  terms  dipeptids;  such,  for  example,  are  glycyl- 
glycin,  alanyl-alanin  and  leucyl-leucin;  chains  consisting  of  three 
links  he  terms  tripeptids,  such  being,  for  example,  diglycyl- 
glycin,  and  leucyl-glycyl-glycin;  chains  consisting  of  four  links 
are  termed  tetrapeptids,  and  so  on,  the  higher  members  of  the 
series  being  collectively  termed  poly  peptids. 

The  surpassing  interest  of  these  investigations  lies  in  the  fact 
that  Fischer  considers  many  of  his  polypeptids  to  be,  in  all  proba- 
bility, identical  with  some  of  the  natural  peptones  and  sub- 
peptones;  while  others  probably  merit  inclusion  among  the, 
proteins  themselves.  Thus  the  octadecapeptid  1-leucyl-triglycyl- 
1-leucyl-triglycyl-l-leucyl-octaglycyl-glycin,  and  the  tetradeca- 
peptid  1-leucyl-triglycyl-l-leucyl-octaglycyl-glycin  so  closely  re- 
semble, in  general  properties,  the  ordinary  proteins,  that,  as 
Fischer  puts  it,  they  would  have  been  classed  as  proteins  had 
they  been  first  met  with  in  nature  (18)  (19).  Thus  they  give 
the  biuret  reaction,  form  opalescent  watery  solutions,  and  the 
tetradecapeptid  is  precipitated  by  ammonium  sulphate,  by  tannic 
acid  and  by  phosphotungstic  acid.  As  they  do  not  contain 
tyrosin,  tryptophane  or  cystin  they  fail  to  give  such  protein 
color  reactions  as  depend  upon  the  presence  of  these  groups. 
The  molecular  weight  of  the  octadecapeptid  is  1213,  and  the 


14  CHEMICAL  STATICS 

substitution  of  phenylalanin,  tyrosin  and  cystin  in  the  place  of 
glycin  groups  would  increase  this  weight  two  or  three  times, 
giving  a  value  which  is  of  the  same  order  as  that  of  the  more 
modern  estimations  of  the  (minimal)  molecular  weights  of  many 
of  the  natural  proteins.* 

A  whole  series  of  the  polypeptids  give  the  typical  peptone 
biuret  reaction,  and  such  as  contain  tyrosin  give  also  Millon's 
reaction.  The  biuret  reaction  is,  with  the  glycin  compounds, 
first  encountered  in  the  tetrapeptid,  but  it  is  given  by  other 
tripeptids.  It  is  more  intense  the  greater  the  length  of  the  poly- 
peptid  chain,  and  it  is  also  intensified  by  the  carboxyl  group  or 
by  conversion  of  the  carboxyl  group  into  an  acid  amide  group. 
The  majority  of  the  polypeptids  are  readily  soluble  in  water, 
and  such  as  are  with  difficulty  soluble  in  water  are  readily  soluble 
in  dilute  mineral  acids  and  alkalies  with  which  they  combine; 
they  are  less  soluble  in  solutions  of  acetic  acid.  As  a  rule  they 
are  insoluble  in  absolute  alcohol,  but  in  alcohol  containing  a 
little  watery  ammonia  they  are  generally  soluble;  on  boiling  off 
the  ammonia  they  are  precipitated  again.  Under  conditions 
involving  dehydration,  e.g.,  heating  or  treatment  of  the  esters 
with  alcoholic  ammonia,  the  dipeptids  are  converted  into  diketo- 
piperazines  which  are  ring-compounds.  Under  similar  conditions 
the  polypeptids  are  modified  in  an  analogous  manner,  with  the 
formation  of  ring-compounds. 

Upon  hydrolysis  the  peptids  break  down  into  their  constituent 
amino-acids,  the  imino  groups  in  the  polypeptid  molecule  being 
converted,  by  the  taking  up  of  water,  into  amino  groups.  A 

»  *  Emil  Fischer  strongly  inclines  to  the  opinion  that  the  higher  estimates, 
such  as  12,000  and  15,000  for  the  minimal  weight  of  the  protein  molecule, 
which  are  freely  cited  in  the  older  literature  upon  proteins,  are  in  error,  since 
the  admixture  of  a  small  quantity  of  another  protein  might  easily  raise  the 
calculated  value  to  this  magnitude,  and  we  have  no  proof  of  the  chemical 
individuality  of  the  majority  of  our  protein  preparations,  even  when,  as  some- 
times happens,  they  are  crystallizable.  An  exception  appears  to  be  afforded 
by  haemoglobin,  the  minimal  weight  of  which  is  placed  by  many  observers, 
employing  adequate  chemical  and  physico-chemical  technique,  at  or  in  the 
neighborhood  of  16,000.  But  then  haemoglobin  is  probably  to  be  regarded  as 
a  salt-like  compound  of,  possibly,  two  or  more  molecules  of  a  basic,  histone- 
like  protein  (globin)  with  a  non-protein  acid,  namely  hsematin.  In  a  similar 
way,  we  shall  see  (Chaps.  V  and  IX)  that  casein,  which  is  possessed  of  a 
minimal  molecular  weight  of  4000-4400,  forms,  under  certain  conditions,  salts 
of  which  the  molecular  weight  is  double  or  four  times  this. 


OCCURRENCE  OF  PEPTIDS  15 

very  large  number  of  them  are  hydrolysed  by  the  proteolytic 
enzymes,  pepsin,  trypsin,  etc.,  and  in  some  cases,  at  all  events, 
it  is  certain  that  the  hydrolysis  takes  place  in  stages,  as  it  does 
with  the  proteins  and  peptones  (1).  Further  discussion  of  the 
mode  of  hydrolysis  of  the  polypeptids  by  enzymes  will  be  found 
in  the  chapters  dealing  with  the  chemical  dynamics  of  protein 
systems. 

5.  The  Occurrence  of  Peptids  among  the  Products  of  Protein 
Hydrolysis.  —  It  is  highly  probable  that  many  of  the  bodies 
which  are  known  to  the  biochemist  as  "proteoses"  "  peptones, " 
etc.,  will  turn  out  to  be  identical  with  some  of  the  polypeptids 
already  synthesized;  this  identity  has,  however,  not  yet  been 
proven.  But  several  of  the  simpler  peptids^and  one  tetrapeptid 
have  already  been  isolated  from  protein  digests.  These  are  to 
be  regarded  as  products  of  incomplete  hydrolysis,  intermediate 
between  the  higher  complexes  and  the  simple  amino-acids  which 
result  from  their  complete  decomposition. 

The  presence  of  a  dipeptid  amongst  the  products  of  the  hy- 
drolysis of  a  protein  of  silk,  silk-fibroin,  was  detected  by  Fischer 
and  Bergell  in  1902  (16).  Later,  Fischer  and  Abderhalden  (9) 
showed  that  its  anhydride,  which  they  isolated,  was  the  diketo- 
piperazine  of  glycyl-alanin,  and  that  it  could  not  possibly  have 
arisen,  by  synthesis  from  glycin  and  alanin,  during  the  process 
of  its  isolation.  At  the  same  time  they  isolated  another  dipep- 
tid from  among  the  products  of  the  incomplete  hydrolysis  of 
silk-fibroin,  namely  glycyl-1-tyrosin,  while  from  among  the  prod- 
ucts of  the  incomplete  hydrolysis  of  elastin  glycyl-1-leucin  was 
obtained.  Later,  Fischer  and  Abderhalden  (22),  by  partial 
hydrolysis  of  the  silk-fibroin,  obtained  a  peptone-like  substance, 
precipitable  by  phosphotungstic  acid,  easily  soluble  in  water, 
insoluble  in  alcohol,  precipitable  from  its  aqueous  solution  by 
saturation  with  ammonium  sulphate  or  sodium  chloride,  which 
proved  to  be  a  tetrapeptid,  yielding,  on  hydrolysis,  two  molecules 
of  glycin,  one  of  alanin  and  one  of  tyrosin.  Its  molecular  weight, 
determined  by  the  cryoscopic  method,  was  350.  The  synthetic 
pentapeptid,  1-leucyl-triglycyl-l-tyrosin,  possesses  very  similar 
properties,  so  that  the  peptones  are  not  necessarily  exceedingly 
complex  substances,  nor  is  excessive  complexity  necessary  in 
order  that  substances  of  this  type  may  be  precipitable  from  their 
aqueous  solutions  by  saturation  with  ammonium  sulphate. 


16  CHEMICAL  STATICS 

A  number  of  other  peptids  have  been  isolated  by  various 
observers  (42)  (48)  from  among  the  products  of  the  incomplete 
hydrolysis  of  proteins. 

6.  The  Analysis  and  Characterization  of  Proteins  by  the  Deter- 
mination of  the  Chemical  Groups  Characteristic  of  the  Different 
Ammo-acids.  —  The  hydrolysis  of  the  proteins  is  accompanied 
by  a  very  marked  increase  in  the  total  number  of  free  amino 
groups.  This  is  due  to  the  fact,  now  incontestibly  established, 
that  the  various  amino-acid  radicals  of  the  protein  molecule 
are  attached  to  one  another  in  an  end  to  end  or  catenary  linkage, 
through  the  union  of  the  amino  group  of  one  amino-acid  with  the 
carboxyl  group  of  the  adjacent  acid,  in  accordance  with  the 
general  equation: 

HaNRiCOOH+H.HNRaCOOH  =  HaNRiCOHNR^COOH+HaO. 

The  reversion  of  this  reaction,  in  hydrolysis,  leads,  of  course,  to 
the  transformation  of  an  imino  or  potentially  imino  group  into 
a  free  amino  group  and  the  series  of  such  transformations  which 
constitutes  the  process  of  the  hydrolysis  of  a  protein  leads  to  the 
appearance  of  a  large  number  of  free  amino  groups  which  were 
not  present  as  such  in  the  unhydrolysed  protein  molecule. 

Free  amino  groups  in  the  aliphatic  series  have  the  well-known 
property  of  reacting  with  nitrous  acid  with  the  liberation  of 
nitrogen,  in  accordance  with  the  equation: 

RNH2  +  HN02  =  ROH  +  N2  +  H2O. 

Very  ingenious  advantage  has  been  taken  of  this  fact  by  D.  D. 
Van  Slyke  (69)  (70)  (2)  in  the  method  which  he  has  devised  and 
which  is  now  very  widely  employed  for  the  determination  of  the 
distribution  and  partition  of  nitrogen  within  the  protein  molecule. 

This  method  consists  essentially  in  the  following  process.  The 
protein  having  been  in  the  first  place  subjected  to  complete 
hydrolysis,  the  ammonia  in  the  mixture  of  products  (derived 
from  "amid"  nitrogen  in  the  protein  molecule)  is  first  removed 
by  vacuum  distillation  and  separately  determined.  The  residual 
mixture  of  products  is  then  treated  with  phosphotungstic  acid, 
which  results  in  the  precipitation  of  the  diamino-acids,  namely 
cystin,  arginin,  lysin  and  histidin.  A  determination  of  sulphur 
yields  a  measure  of  the  cystin-content.  Arginin  has  the  property 
of  yielding  one-half  of  its  nitrogen  in  the  form  of  ammonia  on 


TYPES  OF  UNION  17 

boiling  with  alkali.  The  quantity  of  ammonia  developed  on 
boiling  the  precipitate  with  alkali  therefore  affords  a  measure  of 
the  content  of  arginin.  The  total  nitrogen  in  the  precipitate 
is  now  determined  and  from  it  is  subtracted  the  proportion  of 
nitrogen  which  is  contributed  by  the  cystin  and  arginin  content. 
The  residual  nitrogen  is  derived  from  lysin  (=  x)  and  histidin 
(=  y).  On  treatment  with  nitrous  acid  lysin  yields  a  volume 
of  free  nitrogen  corresponding  to  the  whole  of  its  nitrogen 
content  (=  x),  while  histidin  yields  a  volume  of  free  nitrogen 
which  corresponds  to  two-thirds  of  its  nitrogen  content  (=  f  y). 
The  amino-nitrogen  content  of  the  precipitate  is  therefore  deter- 
mined by  the  nitrogen  yield  on  treatment  with  nitrous  acid  and 
after  subtraction  of  the  amino-nitrogen  contents  of  arginin 
(=  one-fourth  of  its  total  nitrogen)  and  of  cystin  (=  the  whole 
of  its  nitrogen  content),  the  residual  amino  nitrogen  evidently 
represents  the  whole  of  the  lysin  nitrogen  plus  three-fourths 
of  the  histidin  nitrogen.  But  the  determination  of  the  total 
nitrogen  in  the  precipitate  and  the  subtraction  therefrom  of  the 
cystin  and  arginin  nitrogen  has  already  given  us  a  measure  of 
the  total  nitrogen  yielded  by  the  lysin  and  histidin.  Subtract- 
ing, therefore,  the  amino-nitrogen  yield  of  these  amino-acids 
the  difference  evidently  corresponds  to  one-third  of  the  histidin 
nitrogen,  from  which  the  contents  of  histidin  and  lysin  may 
readily  be  computed. 

jr.  In  the  filtrate,  after  the  separation  of  the  diamino-acid  precipi- 
tate, the  total  nitrogen  and  the  amino-nitrogen  are  separately 
determined.  The  difference  yields  a  measure  of  the  nitrogen 
contained  in  pyrrollidine  (prolin  and  oxyprolin)  or  indol  (trypto- 
phane)  rings. 

,  During  the  hydrolysis  by  hydrochloric  acid  a  small  amount 
of  a  very  deeply  colored  precipitate  separates  out.  The  nitro- 
gen content  of  this  precipitate  is  the  so-called  "  melanin "  or 
"humin"  nitrogen.  According  to  Gortner  and  Blish  (27)  this 
is  derived  from  a  portion  of  the  tryptophane  and,  in  the  presence 
of  a  sufficiency  of  carbohydrate,  the  yield  of  melanin  nitrogen 
is  a  quantitative  measure  of  the  tryptophane  content  of  the 
protein. 

7.  Types  of  Union  in  the  Protein  Molecule.  —  Following  the 
recognition  of  the  fact  that  the  proteins  are  complexes  built  up 
by  the  union  of  amino-acids,  the  question  of  the  mode  of  union 


18  CHEMICAL  STATICS 

between  them  became  one  of  paramount  importance.  Hof- 
meister  (30)  has  pointed  out  that  it  is  possible  to  conceive  of 
several  ways  in  which  the  acids  may  be  linked  together  such  as: 

A.  Direct  union  of  the  carbon  atoms,  as: 

I       I 
-C-C- 

I       I 

under  which  condition  the  molecule  would  be  an  immense  chain 
of  carbon  atoms,  and  would  not  be  readily  hydrolysable,  as  the 
proteins  are,  into  its  constituent  amino-acids. 

B.  Ether-like  unions,  as : 

I  I 

-C-O-C- 

I          I 

Such  unions,  however,  would  only  be  possible  when  one  of  the 
two  amino-acids  contained  a  hydroxyl  group.  Only  tyrosin, 
serin  and  oxyprolin,  among  the  amino-acids  which  occur  in  the 
proteins  contain  such  groups,  however,  therefore  this  mode  of 
union  cannot  be  of  general  occurrence  in  the  proteins. 

C.  The  carbon  atoms  may  be  united  by  a  nitrogen  atom,  as: 

I       I       I 
-C-N-C- 

I  I 

Several  varieties  of  this  mode  of  union  are  possible,  such  as: 
-CH2-NH-CH2-    -CH2-NH-CO-    -CH2-NH-C(NH) 
I  II  III 

The  syntheses  accomplished  by  Emil  Fischer,  which  I  have 
described  above,  the  occurrence  of  peptids  among  the  products 
of  the  partial  hydrolysis  of  proteins,  and  their  digestibility  by 
proteolytic  enzymes,  demonstrate  that  type  II  is  the  most  gen- 
eral mode  of  union  among  the  amino-acids  which  constitute 
the  protein  molecule.  The  fact  that  the  proteins  give  the  biuret 
reaction  also  supports  this  view.  It  has  been  shown  by  Schiff  (61) 
that  only  those  substances  which  contain  two  —  CO  —  NH  —  groups, 
or  two  —  CS — NH — ,  or  two  C  (NH)  —  NH  groups,  or,  under  certain 
conditions,  two  —  CH— NH—  groups  yield  the  biuret  test.  That 
such  groups  as: 

I 
-COHN-C-COHN- 

I 


TYPES  OF  UNION  19 

occur  in  the  protein  molecules,  as  they  do  in  the  molecules  of  the 
polypeptids,  is  therefore  highly  probable  and  is  furthermore 
confirmed  by  the  extreme  paucity  of  free  amino  groups  in  the 
native  protein  molecule. 

The  content  of  free  amino  groups  in  the  native  protein  molecule 
may  be  determined  either  by  the  yield  of  nitrogen  on  treatment 
with  nitrous  acid  (43)  (69)  (38)  (71)  or  by  the  method  of  titra- 
tion  with  formaldehyde,  originally  proposed  by  Sorensen  (66) 
(67)  (46)  (38)  which  latter  method  also  enables  us  to  indirectly 
estimate  the  free  carboxyl  groups.  An  examination  of  the  vari- 
ous proteins  by  either  of  these  methods  reveals  the  fact  that  the 
content  of  free  amino  groups  in  the  unhydrolysed  protein  mole- 
cule is  very  small  indeed.  Thus  Van  Slyke  and  Birchard  have 
obtained  the  following  results  (71) : 

PERCENTAGE  OF  TOTAL  NITROGEN  PRESENT  IN  FREE  AMINO  GROUPS 

Haemoglobin 6.0 

Casein 5.5 

Hsemocyanin 4.3 

Gelatin 3.1 

Edestin 1.8 

Gliadin 1.1 

Zein 0.0 

Heteroalbumose 8.1 

Protoalbumose 9.9 

On  the  other  hand,  edestin,  after  complete  hydrolysis  by  hydro- 
chloric acid,  yields  a  volume  of  free  nitrogen,  on  treatment  with 
nitrous  acid,  corresponding  to  no  less  than  79  per  cent  of  its 
total  nitrogen  content  (69).  The  investigations  of  Kossel  and 
Gavrilov  and  of  Van  Slyke  and  Birchard  have  in  fact  shown 
that  the  free  amino  nitrogen  in  the  unaltered  protein  molecule 
exactly  corresponds  in  quantity  with  one-half  the  lysin  nitrogen. 
Hence  zein,  which  contains  no  lysin  (49)  yields  no  free  nitrogen 
on  treatment  with  nitrous  acid.  The  period  required  for  complete 
interaction  of  proteins  with  nitrous  acid  (30  minutes)  is  longer 
than  that  required  by  the  a-amino  groups  (3  to  4  minutes),  but 
corresponds  to  that  found  for  the  co-amino  group  of  lysin.  From 
these  facts  Van  Slyke  and  Birchard  infer  that  one  of  the  two 
amino  groups  of  lysin,  the  o>  group,  exists  free  in  the  protein 
molecule,  and  that  this  group  represents,  within  at  most  a  frac- 
tion of  a  per  cent  of  the  total  protein  nitrogen,  the  entire  amount 


20  CHEMICAL  STATICS 

of  the  free  amino  nitrogen  determinable  in  the  native  proteins 
by  the  nitrous  acid  method.  The  a-amino  groups  which  con- 
stitute the  remaining  and  greater  part  of  the  free  amino  nitrogen 
found  after  complete  hydrolysis  are,  in  the  intact  protein  mole- 
cule, practically  all  condensed  into  peptid  linkings. 

In  the  primary  albumoses  or  first  split-products  of  protein 
hydrolysis,  the  relations  are  different.  The  free  amino  groups 
in  hetero-  and  protoalbumose  exceed  one-half  the  content  of 
lysin  nitrogen  by  3.0  and  4.8  per  cent  of  the  total  nitrogen  re- 
spectively, indicating  that  an  appreciable  proportion  of  the 
a-amino  groups  are  uncovered  in  even  the  first  steps  of  hydrolysis. 

8.  Some  Consequences  of  the  Polypeptid  Structure  of  the 
Protein  Molecule.  —  The  polypeptids  are  as  essentially  amino- 
acids  as  the  amino-acids  out  of  which  they  are  built  up.  Thus 
glycyl-glycin  is  as  typically  an  amino-acid  as  glycocoll  itself, 
since  it  possesses  an  —  NH2  group  as  well  as  a—  COOH  group 
and  for  this  reason  is  presumably  capable  of  forming  compounds, 
not  only  with  acids  and  bases,  but  also  with  neutral  salts  (52). 
On  undergoing  electrolytic  dissociation  it  may  be  supposed  to 
yield  either  hydrogen  (H+)  ions,  or  hydroxyl  (OH7)  ions,  owing 
to  the  occurrence  of  a  reaction  with  water  of  the  type: 

NH3OH 


/ 

R/ 
COOH  XCOOH 

just  as  ammonia,  in  aqueous  solution,  partially  reacts  with  water 
to  form  NH4OH. 

It  is  usually  conceded  that  these  elements  in  the  structure  of 
the  proteins  afford  an  explanation  of  the  power  which  they  pos- 
sess of  neutralizing  both  acids  and  bases,  in  other  words  the 
"amphoteric"  character  of  the  proteins.  To  this  opinion  I  have 
also  formerly  inclined,  but  an  accumulation  of  data  irreconcil- 
able with  this  view  induced  me  some  years  ago  (57)  to  abandon 
it.  Since  that  time  evidence  of  a  perfectly  conclusive  character 
has  been  obtained  and  we  may  now  regard  it  as  an  established 
fact  that  some  elements  in  the  protein  molecule  other  than  ter- 
minal —  NH2  or  —  COOH  groups  are  responsible  for  the  acid-  and 
base-neutralizing  power  which  is  possessed  in  such  a  marked 
degree  by  many  proteins  (vide  Chapters  IV  and  V). 

In  the  first  place,  the  investigations  of  Levites  and  D.  D.  Van 
Slyke,  referred  to  above,  have  shown  that  only  a  very  small 


CONSEQUENCES  OF  THE  POLYPEPTID  STRUCTURE      21 

proportion  of  the  nitrogen  in  proteins  is  present  within  their 
molecules  in  the  form  of  —  NH2  groups.  Thus  in  the  case  of 
edestin,  as  we  have  seen,  only  1.8  per  cent  of  the  total  nitrogen 
is  present  in  the  form  of  free  —  NH2  groups. 

Now  edestin,  as  Osborne  (47)  has  shown,  is  insoluble,  when 
in  the  free  condition,  in  water.  It  forms  an  insoluble  hydro- 
chloride  containing  14  X  10~5  equivalents  of  HC1  per  gram 
and,  on  further  addition  of  acid,  passes  into  solution.  Its  com- 
bining-capacity  for  acids  does  not  remain  constant,  however,  for 
at  neutrality  to  tropseolin,  which  corresponds  to  a  free  acidity 
of  from  0.01  to  0.001  H+  (60)  it  neutralizes  127  X  10~5  equiva- 
lents of  acid  per  gram.  Hence,  if  the  acid  is  neutralized  by 

—  NH2  groups  of  edestin,  the  number  of  these  groups  in  each 
molecule  of  edestin  must  be  at  least  *££-  =  9.*    From  the  former 
determination   it   would   appear   that   the  molecular   weight   of 
edestin  is  7000,  and  this  corresponds  with  the  molecular  weight 
indicated  by  its  tyrosin  and  glutamic  acid  content  (40)  (1  mol. 
tyrosin  +  3  mols.  glutamic  acid  +•••)•     Nine  —  NH2  groups 
in  this  molecule  would  correspond  to  over  ten  per  cent  of  the 
total  nitrogen,  or  almost  exactly  to  the  whole   —  NH2  content 
of  the  arginin,   calculated  as  the  free  diamino-acid,  which  the 
edestin  molecule  contains.     Since  only  1.8  per  cent  of  the  total 
nitrogen  of  the  edestin  molecule  is  present  therein  in  the  form 
of  —  NH2  groups  no  less  than  eighty  per  cent  of  the  neutralizing 
power  of  the  edestin  for  acids  must  be  accounted  for  in  some 
other  fashion  than  by  the  assumption  of  a  union  of  the  acid  with 
free  —  NH2  groups. 

From  the  investigations  of  Erb  (14),  although  the  exact  in- 
terpretation' of  his  results  is  in  some  respects  open  to  question 
(Chap.  IV),  it  would  appear  that  the  combining-weight  of  egg 
albumin  for  acids  may  be  as  low  as  152,  while  its  molecular  weight 
is,  according  to  Hofmeister,  5400  or  some  multiple  of  this.  Hence 
upon  the  assumption  that  terminal  —  NH2  groups  bind  these 
acids,  there  must  be  at  least  35  of  them  in  egg  albumin  (45), 
which  would  correspond  to  no  less  than  69  per  cent  of  the  total 
nitrogen  in  this  protein. 

*  Osborne  believes  that  an  insoluble  "monochlorhydrate"  is  also  formed, 
containing  7  X  10"6  equivalents  per  gram  which  would  raise  the  number  of 

—  NH2  groups  to  18,  but  would,  at  the  same  time,  double  the  estimate  of  the 
molecular  weight. 


22  CHEMICAL  STATICS 

It  has  been  pointed  out  by  Kossel  and  Cameron  (37)  that  the 
acid-combining  capacity  of  the  protamin,  salmin,  is  equal  to  the 
combining  capacity  of  the  guanidin  groups  of  the  arginin  radicals 
which  it  contains,  yet  salmin  yields  no  nitrogen  on  treatment 
with  nitrous  acid.  Sturin  (41)  contains  67  per  cent  of  its  nitro- 
gen in  the  form  of  arginin,  10  per  cent  in  the  form  of  histidin 
and  6  to  7  per  cent  in  the  form  of  lysin.  It  yields  nitrogen  on 
treatment  with  nitrous  acid  corresponding  to  the  co-amino  group 
of  the  lysin.  Only  about  three  out  of  every  hundred  nitrogen 
atoms  in  sturin  are  therefore  present  in  the  form  of  free  —  NH2 
groups.  Yet  one  hundred  nitrogen  atoms  in  sturin  will  neu- 
tralize no  less  than  24  equivalents  of  acid.  All  of  these  pro- 
tamins,  moreover,  possess  a  certain,  although  relatively  inferior 
power  of  neutralizing  bases. 

The  number  of  terminal  —  COOH  groups  in  any  protein  can- 
not be  much  in  excess  of  the  terminal  —  NH2  groups,  as  the  formol- 
titration  and  behavior  towards  cupric  hydroxide  (32)  (33)  (66) 
(67)  show,  and  also  because  the  protein  would  otherwise  be 
overwhelmingly  acid  in  character  (30)  and  the  majority  of  the 
proteins  possess  a  distinct  capacity  for  neutralizing  acids,  even 
when  they  are  themselves  predominantly  acid.  Now  free  casein  * 
is  insoluble  in  water,  but  when  combined  with  acids  or  with 
bases  it  is  soluble.  To  carry  one  gram  of  casein  into  solution 
11.4  X  10~5  equivalents  of  base  just  suffice,  indicating  a  com- 
bining-weight  for  casein  of  about  8800.  The  tyrosin,  glutamic 
acid  and  sulphur  contents  of  casein  indicate  a  minimal  molecu- 
lar weight  of  from  4000  to  4400. 

In  the  presence  of  excess  of  base,  however,  casein  attains  a 
maximal  combining  capacity  (measured  by  the  gas-chain)  of 
180  X  10~5  equivalents  per  gram,  so  that  it  behaves  like  a  16- 
basic  acid,  and  if  —COOH  groups  bind  the  base  there  must 
be  16  of  them  in  the  molecule,  corresponding  to  25  per  cent  of 
the  total  oxygen,  or,  almost  exactly,  to  the  percentage  of  the  total 
oxygen  which  is  contained  in  the  —COOH  groups  of  the  glutamic 
add  in  the  casein  molecule,  calculated  as  the  free  acid.  In  order 
to  provide  so  many  free  carboxyls,  the  form  of  the  casein  mole- 
cule would  necessarily  be  that  of  a  branched  chain,  or  the  radi- 
ating spokes  of  a  wheel,  at  the  centre  of  which  must  exist  unions 
of  the  type:  — C  —  C— 

*  For  references  to  the  sources  of  these  data  Cf.  Chap.  IV. 


CONSEQUENCES  OF  THE  POLYPEPTID  STRUCTURE      23 

and  the  regular  decomposition  of  the  proteins  into  their  con- 
stituent amino-acids,  upon  hydrolysis,  would  be  unintelligible 
(30).  Moreover,  in  the  synthetical  polypeptids,  which  closely 
resemble  the  natural  peptones  in  their  behavior,  the  linkage  of 
the  amino-acids  is  not  radial  but  catenary  in  character*  and 
the  peptids  which  have  been  isolated  from  the  mixed  products 
of  partial  protein  hydrolysis  are  likewise  catenary  in  structure. 

In  the  second  place,  as  Vernon  has  pointed  out  (72),  although 
the  power  of  the  sum  of  the  decomposition-products  of  a  protein 
to  neutralize  bases  is  greater,  yet  it  is  only  very  slightly  greater 
than  that  of  the  unhydrolysed  protein.  Now  in  the  process  of 
hydrolysis  the  —  COHN—  groups  of  the  protein  are  split  into 
—  NH2  and  —  COOH  groups;  yet  this  results  in  no  pronounced 
gain  of  combining-capacity  for  bases.  The  obvious  conclusion  is 
that  the  —COHN—  group  within  the  protein  molecule  must  be 
nearly  as  efficient  in  accomplishing  the  neutralization  of  bases 
as  the  —COOH  group  of  the  constituent  amino-acids  out  of 
which  the  protein  is  built  up. 

Direct  proof,  however,  that  terminal  —  NH2  groups  are  not 
responsible  for  any  appreciable  proportion  of  the  acid-combining 
capacity  of  proteins  has  been  furnished  by  the  recent  experiments 
of  Blasel  and  Matula  (5)  and  Pauli  and  Hirschfeld  (51).  These 
investigators  prepared  deaminized  gelatin  by  acting  upon  gelatin 
with  nitrous  acid,  thus  destroying  all  the  free  —  NH2  groups  in 
the  molecule.  They  then  compared,  with  the  aid  of  the  gas- 
chain,  the  acid-combining  capacity  of  the  deaminized  gelatin 
with  that  of  normal  gelatin.  They  found  that  the  combining- 
capacity  of  deaminized  gelatin  for  acids  is  but  slightly  inferior 
to  that  of  ordinary  gelatin,  indicating,  beyond  any  question,  that 
the  combining-capacity  of  gelatin  for  acids  is,  in  very  large  pro- 
portion, attributable  to  elements  of  the  molecule  other  than 
free  —  NH2  groups.  The  inference  is  unavoidable  that  the  ele- 
ments of  the  molecule  which  actually  participate  in  the  union 
with  acids  are,  in  very  large  proportion,  the  —COHN—  groups 
within  the  body  of  the  protein  molecule. 

Very  strong  evidence  that  the  same  structural  elements  of  the 
protein  molecule  are  responsible  for  the  neutralization  of  bases 
by  proteins  has  also  been  recently  afforded  by  the  investigations 

*  Even  when  dicarboxylic  acids  enter  into  the  compounds,  Cf .  Fischer  and 
Koenigs  (24),  and  Fischer  and  Schmidlin  (26). 


24  CHEMICAL  STATICS 

of  Osborne  and  Leavenworth  (50)  who  have  shown  that  edestin 
combines  with,  or  holds  in  solution,  34.67  per  cent  of  its  weight 
of  copper  in  the  form  of  cupric  hydroxide.  This,  if  we  assume 
that  each  copper  atom  unites  with  one  nitrogen  atom,  involves 
the  union  of  cupric  hydroxide  with  ten  out  of  every  sixteen  atoms 
of  nitrogen  in  the  edestin  molecule.  Now  this  is  exactly  the 
proportion  of  nitrogen  which  edestin  yields  in  the  form  of  amino- 
nitrogen  after  complete  hydrolysis.  In  other  words  it  is  exactly 
equal  to  the  proportion  of  —  COHN—  groups  which  the  unhydro- 
lysed  molecule  contains.  Precisely  similar  results  were  obtained 
with  gliadin. 

To  account  for  the  high  acid-  and  base-combining  capacity  of 
the  proteins  we  must  therefore  look,  not  to  the  terminal  —  NH2 
or  —  COOH  groups,  but  to  the  —COHN—  groups  within  the  body 
of  the  protein  molecule.  Now  two  varieties  of  this  union  can  be 
conceived,  between  which  it  has  not  proved  possible  as  yet  to 
decide  by  any  direct  method  of  analysis.  Thus  glycyl-glycin 
may  conceivably  be  either: 

Keto-form 
H2N.CH2.CO  -  HN.CH2.COOH 

or  Enol-form 

H2N.CH2.C(OH)  =  N.CH2.COOH 

and  our  analytical  data,  and  the  modes  of  decomposition  and 
synthesis  of  the  proteins  and  peptids  do  not  enable  us  with  any 
degree  of  certainty  to  distinguish  between  them.  Neither  form 
is  therefore  inconsistent  with  our  present  knowledge  of  the  syn- 
thesis and  hydrolysis  of  proteins  and  polypeptids,  but  while 
the  keto-form  of  the  —COHN—  group  would  conceivably  possess 
the  power  of  neutralizing  acids  it  offers  no  probable  point  of  union 
for  bases.  The  enol-form,  on  the  contrary,  would  provide  a 
point  of  union  for  either  acids  or  bases. 

According  to  Werner's  theory  of  valencies  the  nitrogen  in 
either  of  these  unions  contains  two  latent  valencies,  positive  and 
negative,  which,  while  the  nitrogen  is  trivalent,  neutralize  one 
another,  but  which,  when  .the  nitrogen  becomes  pentavalent  are 
capable,  respectively,  of  neutralizing  a  negative  and  a  positive 
radical.  The  enol  type  of  union  carries  with  it  the  possibility 
of  the  following  types  of  reaction : 


CONSEQUENCES  OF  THE  POLYPEPTID  STRUCTURE      25 

H 

I 
-COH=N-  +  Na+  +  OH'  =  -CONa++  +  ^N-          (i) 

I 

OH 
and 

H 


-COH  =  N-  +  H++C1'  =  -COH+++  ^N-         (ii) 

I 

Cl 
yielding,  in  each  case,  only  protein  ions. 

I  have  already  incidentally  dwelt  upon  the  fact,  in  connection 
with  Kossel's  theory  of  a  protamin  nucleus  of  proteins  and  in 
connection  with  the  neutralizing  powers  of  edestin  for  acids  and 
of  casein  for  bases,  that  there  is  reason  to  suspect  that  diamino 
and  dicarboxylic  radicals  in  the  protein  molecule  play  a  pre- 
dominant part  in  accomplishing  the  neutralization  of  acids  and 
bases,  and  electrochemical  data,  as  we  shall  see,  lend  further 
support  to  this  view.  Accordingly,  the  above  formulae,  which 
represent  the  reaction  when  only  a  single  —  COHN—  bond  is 
involved  should  probably,  at  least  in  many  instances,  be  doubled, 
and  in  the  following  possibilities  would  then  exist: 
In  combination  with  bases, 

H  OH 
\  / 

.COH.N-  /COK++     ^N- 

R'  +  KOH  +  H20  =  R'  +  (iii) 

XCOH.N-  NCOH++     <*N- 

/  \ 
H     OH 

H    OH 
\  / 

/COH.N-  /COK++     ^N- 

R'  +2KOH  =  R^  +  (iv) 

XCOH.N-  XCOK++     ^N- 

/  \ 
H    OH 
In  combination  with  acids, 

H    Cl 

\  / 
-COH.N.  -COH++     ^Nv 

)R  +  HC1  +  H20=  +        )R        (v) 

-COH.NX  -COH++     ^Nx 

/  \ 
H    OH 


26  CHEMICAL  STATICS 

H    Cl 
\  / 
-COH.N.  -COH++     ^N. 

/  R  +  2  HC1  =  -f        ^  R        (vi) 

-COH.NX  -COH++     ^Nx 

H     Cl 

It  is  obvious  that  in  reactions  (iii)  and  (v)  the  molecules  of 
water  may  or  may  not  participate  in  the  reaction;  also  that  the 
anionic  groups  in  reactions  (iii)  and  (iv)  may  or  may  not  be 
united  to  form  a  single  quadrivalent  cation  (derived  from  a 
dicarboxylic  acid  group).  As  we  shall  see  (Chapter  X.),  no  evi- 
dence has  been  found  (at  least  among  the  compounds  of  casein 
or  of  serum  globulin  with  bases)  of  the  occurrence  of  reactions 
of  the  type  represented  by  equation  (iii).  Equation  (iv)  faith- 
fully represents,  so  far  as  the  electrochemical  data  are  concerned, 
the  mode  of  combination  of  bases  with  these  proteins,  and  the 
anionic  groups  are  probably  united  to  form  one  quadrivalent 
union.  The  union  of  serum  globulin  and  ovomucoid  with  acids 
follows  equation  (v)  (with  or  without  the  molecule  of  water) 
when  the  concentration  of  acid  is  low;  but  at  higher  acidities 
ovomucoid,  at  least,  combines  with  hydrochloric  acid  in  the 
manner  indicated  by  equation  (vi). 

In  the  succeeding  chapters  of  this  work  we  shall  see  that  this 
hypothesis  regarding  the  mode  of  union  between  the  proteins  and 
acids  and  bases  is  supported  by  the  following  facts : 

(i)  The  compounds  which  the  proteins  form  with  acids  and 
bases,  when  dissolved  in  water,  are  excellent  conductors  of  elec- 
tricity, and  true  electrolytes  (65)  (55)  (4),  yet  they  do  not,  for 
example,  yield  chlorine  ions,  when  the  compound  in  question  is  a 
hydrochloric  acid  compound  (8)  (59),  nor  does  the  potassium 
hydrate  compound  yield  potassium  ions,  or  the  calcium  hydrate 
compound  calcium  ions  (56).  The  equivalent  conductivities  of 
the  compounds  at  infinite  dilution  are  such  as  would  indicate 
the  presence  only  of  bulky  organic  ions,  travelling  under  a  given 
fall  of  potential  at  the  constant  minimal  equivalent  velocity  of 
about  20  X  10~5  cm.  per  sec.  per  volt  per  cm.  fall  in  potential 
at  30°  C.  which  is  characteristic  for  such  ions  (6). 

(ii)  Edestin  will  displace  NaOH  from  its  combination  with 
hydrochloric  acid  (T.  B.  Osborne,  Cf.  Chapter  V)  and  casein, 


CONSEQUENCES  OF  THE  POLYPEPTID  STRUCTURE      27 

although  insoluble  in  water,  will  displace  carbonic  acid  from  its 
combination  with  calcium  hydrate  (W.  A.  Osborne,  Cf.  Chap.  V). 
Solutions  of  caseinates  of  the  bases  may  be  obtained  which  are 
pronouncedly  acid  in  reaction  (Robertson,  Cf.  Chap.  V).  Since 
in  all  these  cases  the  molecular  concentration  of  the  protein 
is  very  low,  and  the  compounds  which  are  formed  are  quite 
highly  electrolytically  dissociated  (Cf.  above),  were  the  forma- 
tion of  these  compounds  due  to  the  replacement  of  OH'  groups  of 
—  NH3OH  groups  by  acid  radicals  or  of  H+  groups  in  —  COOH 
groups  by  bases,  then  the  "strength,"  i.e.,  the  degree  of  disso- 
ciation of  edestin  as  a  base  must  be  equal  to  or  greater  than 
that  of  NaOH,  while  that  of  casein  as  an  acid  must  be  much 
greater  than  that  of  H2C03  and  comparable  with  the  degree  of 
dissociation  of  NaOH  at  very  high  dilutions  (since  Na  caseinate 
may  be  prepared  which  is  acid  in  reaction).  Such  conclusions, 
applied  to  bodies  which  are  amphoteric,  are,  of  course,  absurd. 
Were  the  formation  of  potassium  caseinate  due  to  the  formation 
of  a  salt  such  as 

RCOO'  +  K+ 

an  acid  solution  of  this  compound  could  no  more  exist  than  an 
acid  solution  of  potassium  aluminate.  As  in  similar  cases  which 
occur  in  the  domain  of  inorganic  chemistry,  we  can  interpret 
these  phenomena  only  by  assuming  that  the  basic  radical  in  the 
casein  compounds,  and  the  acid  radical  in  the  edestin  compound 
are  bound  up  in  a  non-dissociable  form.  Since  the  casein  com- 
pounds, at  least,  when  in  solution  in  water,  are  notable  conduc- 
tors of  electricity,  they  must  dissociate  at  some  other  point  in 
the  molecule  than  that  of  the  union  between  the  base  and  the 
protein. 

(iii)  Each  equivalent  *  of  a  monobasic  acid  or  monacid  base 
neutralized  by  serum  globulin  or  casein  yields  two  equivalents  of 
the  protein  compound  (Cf.  Chap.  X).  This  obviously  corre- 
sponds with  the  mode  of  dissociation  depicted  above,  while,  if 
terminal  —  NH2  or  —COOH  groups  accomplished  the  union, 
each  equivalent  of  neutralized  acid  or  base  would  produce  only 
one  equivalent  of  salt. 

(iv)  On  successive  additions  of  1,  2,  3,  etc.,  equivalents  of 

*  That  is,  gram-molecule  divided  by  the  valency  of  the  combining  radical, 
in  this  instance  unity. 


28  [CHEMICAL  STATICS 

monacid  bases  to  a  solution  of  an  organic  polybasic  acid  of  the 
type  R(COOH)m,  and  the  formation  of  salts  by  the  replacement 
of  H  atoms  in  the  —COOH  groups,  the  osmotic  pressure  of  the 
solution  would  increase  (provided  the  salts  were  highly  dissociated) 
in  the  same  proportion,  namely  2,  3,  4,  etc.  The  experimental 
fact,  for  casein  (58),  is  that  the  osmotic  pressure  (=  depression 
of  the  freezing-point)  increases  in  the  proportion  2,  4,  6,  etc., 
i.e.,  each  successive  equivalent  of  neutralized  base  gives  rise  to 
an  equal  number  of  ions.  This  obviously  corresponds  with  what 
would  be  expected  were  the  union  and  its  mode  of  dissociation 
of  the  type  outlined  above. 

In  addition  to  these,  a  host  of  minor  details  in  the  behavior 
of  the  protein  salts,  which  would  be  very  hard  to  explain  upon 
any  other  basis,  admit,  as  we  shall  see,  of  a  simple  explanation 
on  the  basis  of  the  hypothesis  outlined  above. 

The  poly-amino-acid  structure  of  the  proteins,  however,  carries 
with  it  other  possibilities  which  are  of  importance  in  interpreting 
their  behavior.  Some  measure  of  the  dissociations 

/NH2 

R'          +H+ 
XCOO' 

and 

,NH2  /NH3+ 

T5    '  I        TT    S~\     "R  I        OTT' 

XCOOH  XCOOH 

must  undoubtedly  occur,  although,  in  the  majority  of  cases  (a 
notable  exception  being  afforded  by  the  protamins)  these  modes 
of  dissociation  must  play  a  very  subordinate  part.  Then  the 
terminal  —  NH2  and  —COOH  groups  (and  at  least  one  of  these 
must  exist  at  either  end  of  a  chain  of  amino-acids)  may  neutralize 
themselves  internally,  thus : 

NH3 

R-COO 

forming  what  Winkelblech  terms  an  " internal  salt"  (73).  Such  a 
molecule,  whatever  the  mode  of  union,  would  of  course  form 
compounds  with  acids  or  bases  with  much  more  difficulty  than 
the  protein  which  had  not  undergone  such  internal  neutralization, 
since,  before  dissociation  could  occur  the  ring-formation  would 


CONSEQUENCES  OF  THE  POLYPEPTID  STRUCTURE      29 

have  to  be  opened  up.     The  internal-salt  formation  may  go  a 
step  further,  with  the  formation  of  anhydrides  such  as 

NH 


such  anhydride  formation  being  frequently  observed  in  the  poly- 
peptids.  Then  two  molecules  of  an  amino-acid,  and  therefore, 
of  a  poly-amino-acid  such  as  protein,  may  unite  with  one  another 
in  either  of  two  ways,  thus  : 

/NH2         HOOC.  /NH.OC.R.NH2 

R'  +  ^R     =    R'  +  H20 

XCOOH         R2^/  NCOOH 

or 

yNH2         HOOCV  /NH.OC, 

R'  +  ^R    =     R'  XR  +  2H20 

XCOOH         HJX'  XCO.HNX 

the  product  being,  in  the  first  instance,  a  poly-amino-acid  of  a 
higher  order  and  greater  molecular  weight,  and  in  the  second 
an  "  internal  salt"  or  anhydride.*  Another  type  of  anhydride 
which  may  be  formed  is  that  of  a  diketopiperazin,  of  the  general 

formula: 

/  CH(R).C(X 
NH/  XNH 


O.CH(R). 


and  these  anhydrides  may  exist  in  two  isomeric  forms,  the  keto 
and  enol  forms,  the  former  being  represented  by  the  above  for- 
mula, and  the  latter  by  the  general  formula  : 


The  enol  form  carrying  with  it  the  possibility  (in  the  presence 
of  water)  of  the  transient  formation  of  salts  with  bases  before  the 
ring  breaks  up.f 

*  Such  as  leucyl-glycin  anhydride. 

t  Cf.  R.  H.  Aders  Plimmer  (53),  Part  2,  p.  36. 


30  CHEMICAL  STATICS 

9.  "Racemized"  Proteins.  —  It  has  recently  been  shown  by 
Kossel  (36)  and  by  Dakin  (11)  (12)  that  treatment  of  proteins 
with  rather  concentrated  alkali  leads  to  a  progressive  diminu- 
tion of  the  optical  rotary  power  of  the  solutions.  This  Dakin 
attributes  to  an  internal  racemization  or,  more  correctly,  enoli- 
zation  of  the  protein  molecule  analogous  to  that  which  occurs  in 
the  hydantoins  (10).  He  depicts  the  reaction  as  follows: 

NH-CO- 
I 
R-CH-CO-NH-CHR-COOH 

NH-CO- 
I 
<=±R-C  =  C(OH)-NH-CHR-COOH 

the  alkali  tending  to  shift  the  equilibrium  towards  the  right. 
The  central  carbon  atom  instead  of  being  attached  by  its  valencies 
to  four  different  groups  is  now  attached  to  three  groups,  to  one 
of  them  by  a  double  bond,  and  any  optical  activity  which  it  may 
have  possessed  owing  to  assymetry  must  have  been  lost. 

The  "racemized"  proteins  thus  prepared  are  totally  resistant 
to  hydrolysis  by  pepsin,  trypsin  or  erepsin.  Putrefactive  bac- 
teria do  not  attack  "racemized"  casein  although  they  slowly 
decompose  the  "racemized"  caseose  (proteose)  which  is  simul- 
taneously formed  through  partial  hydrolysis  of  the  casein  and 
subsequent  "racemization"  of  the  higher  products  of  the  hydroly- 
sis. "Racemized"  egg  albumin  (7)  and  casein  (63)  are  non- 
antigenic.  The  amino-acids  resulting  from  the  complete  hy- 
drolysis of  "racemized"  protein  by  acid  are  for  the  most  part 
optically  inactive  (12). 

A  number  of  objections  have  been  urged  by  Kober,  (32)  against 
Dakin's  interpretation  of  the  progressive  loss  of  optical  activity 
of  proteins  in  alkaline  solutions.  In  the  first  place  he  points 
out  that  the  presence  of  an  oscillating  hydrogen  atom  within 
the  molecule  might  be  anticipated  to  lead  to  the  development  of 
bands  in  the  absorption  spectrum  and  no  absorption-bands  are 
observable  in  protein  solutions  excepting  those  in  the  ultra- 
violet spectrum  which  are  attributable  to  the  phenylalanin  and 
tyrosin  radicals.  This  objection  is  deprived  of  force,  however, 
by  the  fact  that  in  the  hydantoins,  in  which  analogous  "enoli- 
zation"  does  demonstrably  occur,  no  absorption-band  is  developed. 


"RACEMIZED"   PROTEINS  31 

The  objection,  also  raised  by  Kober,  that  racemized  polypetids 
are  hydrolysable  by  trypsin  is  not  valid  because  the  reaction 
depicted  by  Dakin  is  not,  correctly  speaking,  racemization  but 
"  enolization "  and  the  structures  of  polypeptids  formed  from 
racemic  amino-acids  and  the  so-called  " racemized  proteins"  are 
probably  not  in  the  least  analogous.  Finally,  the  fact,  also 
cited  by  Kober  as  an  objection  to  Dakin's  hypothesis,  that  not 
all  of  the  *  amino-acids  resulting  from  the  complete  hydrolysis 
of  enolized  proteins  are  optically  inactive,  merely  tends  to  show 
that  not  all  —  COHN—  groups  are  equally  affected  by  alkali. 

According  to  Underbill  and  Hendrix  (68)  crude  "racemized" 
proteins  produce  toxic  symptoms  when  introduced  into  the  cir- 
culation of  animals.  Purified  "racemized"  proteins  exert  no 
toxic  action.  A  portion,  but  not  all  of  the  toxic  substance  can 
be  removed  from  "racemized"  proteins  by  extraction  with  alcohol. 
Evidently  the  main  reaction  is  complicated  by  the  occurrence 
of  side-reactions  chief  among  which  must  be  hydrolysis  which  is 
rather  rapid  in  solutions  of  the  alkalinity  employed. 

In  terms  of  the  hypothesis  of  protein  ionization  outlined  above 
the  "racemization"  of  proteins  by  alkali  must  consist  in  an  ex- 
change of  hydrogen  atoms  in  the  enol  group  in  accordance  with 
an  equation  such  as: 

NH-CO- 
I 
R-CH-C(OH)  =  N-CH-RCOOH 

NH-CO- 
I 
R_C  =  C(OH)-NH-CH-R-COOH 

with  consequent  transformation  of  the  double  bond  connecting 
carbon  and  nitrogen  in  the  —COHN—  group  into  a  single  bond. 

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p.  294;  54  (1908),  p.  363. 

(2)  Abderhalden,  E.,  and  Van  Slyke,  D.D.,  Zeit.  f .  physiol.  Chem.  74  (1911), 

p.  505. 

(3)  Earth,  L.,  Annalen  der  Chem.  152  (1869),  p.  96. 

(4)  Billitzer,  J.,  Annalen  der  physik.  316  (1903),  pp.  902  and  937. 

(5)  Blasel,  L.,  and  Matula,  J.,  Biochem.  Zeit.  58  (1914),  p.  417. 

(6)  Bredig,  G.  Zeit.  f.  physik.  Chem.  13  (1894),  p.  191. 

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32  CHEMICAL  STATICS 

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p.  51. 

(9)  Curtius,  Th.,  Ber.  d.  d.  chem.  Ges.  16  (1883),  p.  753. 

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(13)  Drechsel,  E.,  Arch.  f.  (Anat.  und)  Physiol.  (1891),  p.  248. 

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(17)  Fischer,  E.,  Ber.  d.  d.  chem.  Ges.  37  (1904),  p.  3062. 

(18)  Fischer,  E.,  Ber.  d.  d.  chem.  Ges.  39  (1906),  p.  2893;  40  (1907),  p.  1754. 

(19)  Fischer,  E.,  Pr.  Chem.  Soc.  23  (1907),  p.  82. 

(20)  Fischer,  E.,  Ber.  d.  d.  chem.  Ges.  41  (1908),  pp.  2860  and  2875. 

(21)  Fischer,  E.,  and  Abderhalden,  E.,  Ber.  d.  d.  chem.  Ges  39  (1906),  p. 

2315. 

(22)  Fischer,  E.,  and  Abderhalden,  E.,  Ber.  d.  d.  chem.  Ges.  40  (1907),  p. 

3544. 

(23)  Fischer,  E.,  and  Fourneau,  E.,  Ber.  d.'d.  chem.  Ges.  34  (1901),  p.  2868. 

(24)  Fischer,  E.,  and  Koenigs,  E.,  Ber.  d.  d.  chem.  Ges.  37  (1914),  p.  4585, 

(25)  Fischer,  E.,  and  Otto,  E.,  Ber.  d.  d.  chem.  Ges.  36  (1903),  pp.  2106  and 

2993. 

(26)  Fischer,  E.,  and  Schmidlin,  J.,  Annalen  der  Chem.  340  (1905),  p.  123 

(Abt.  7). 

(27)  Gortner,  R.  A.,  and  Blish,  M.  J.,  Journ.  Amer.  Chem.  Soc.  37  (1915), 

p.  1630. 

(28)  Grimaux,  Ed.,  C.  R.  Acad.  Sci.  93  (1881),  p.  771;  97  (1883),  pp.  231, 

1336, 1434, 1485, 1540  and  1578.    Rev.  Scient.  (Paris)  1886,  April  18. 

(29)  Hedin,  S.  G.,  Zeit.  f.  physiol.  Chem.  21  (1895),  pp.  155  and  297. 

(30)  Hofmeister,  F.,  Ergeb.  d.  physiol.  1  Abt.  1  (1902),  p.  159. 

(31)  Hufner,  G.,  Zeit.  f.  Chem.  (1868),  p.[391. 

(32)  Kober,  P.  A.,  Journ.  Biol.  Chem.  22  (1915),  p.  433. 

(33)  Kober,  P.  A.,  and  Sugiura,  K,  Journ.  Amer.  Chem.  Soc.  35  (1913),  p. 

1580. 

(34)  Kossel,  A.,  Zeit.  f.  physiol.  Chem.  22  (1896),  p.  176. 

(35)  Kossel,  A.,  Deutsche  Med.  Wochenschr.  (1898),  p.  581;  Zeit.  f.  phy- 

siol. Chem.  25  (1898),  p.  165. 

(36)  Kossel,  A.,  Zeit.  f.  physiol.  Chem.  78  (1912),  p.  402. 

(37)  Kossel,  A.,  and  Cameron,  A.  T.,  Zeit.  f.  physiol.  Chem.  76  (1912),  p.  457. 

(38)  Kossel,  A.,  and  Gavrilov,  N.,  Zeit.  f.  physiol.  Chem.  81  (1912),  p.  274. 

(39)  Kossel,  A.,  and  Kutscher,  F.,  Zeit.  f.  physiol.  Chem.  31  (1900),  p.  165. 

(40)  Kossel,  A.,  and  Patten,  A.  J.,  Zeit.  f.  physiol.  Chem.  38  (1903),  p.  39. 

(41)  Kossel,  A.,  and  Weiss,  F.,  Zeit.  f.  physiol.  Chem.  78  (1912),  p.  402. 

(42)  Levene,  P.  A.,  and  Beatty,  W.  A.,  Ber.  d.  d.  Chem.  39  (1906),  p.  2060. 

(43)  Levites,  S.,  Zeit.  f.  physiol.  Chem.  43  (1904),  p.  202.    Biochem.  Zeit.  20 

(1909),  p.  224. 

(44)  Liebig,  J.,  Annalen  der  Chem.  57  (1846),  p.  127. 

(45)  Mann,  G.,  "Chemistry  of  the  Proteins,"  London  (1906),  p.  147. 


LITERATURE  CITED  33 

(46)  Obermeyer,  F.,  and  Willheim,  R.,  Biochem.  Zeit.  38  (1912),  p.  331. 

(47)  Osborne,  T.  B.,  Journ.  Amer.  Chem.  Soc.  24  (1902),  p.  39. 

(48)  Osborne,  T.  B.,  and  Clapp,  S.  H.,  Amer.  Journ.  Physiol.  18  (1907),  p. 

123. 

(49)  Osborne,  T.  B.,  and  Leavenworth,  C.  S.,  Journ.  Biol.  Chem.  14  (1913), 

p.  481. 

(50)  Osborne,  T.  B.,  and  Leavenworth,  C.  S.,  Journ.  Biol.  Chem.  28  (1916), 

p.  109. 

(51)  Pauli,  W.,  and  Hirschfeld,  M.,  Biochem.  Zeit.  62  (1914),  p.  245. 

(52)  Pfeiffer,  P.,  and  von  Modelski,  J.,  Zeit.  f.  physiol.  Chem.  81  (1912),  p. 

239;  85  (1913),  p.  1. 

(53)  Plimmer,  R.  H.  Aders,  "The  Chemical  Constitution  of  the  Proteins," 

London,  1908. 

(54)  Proust,  M.,  Ann.  de  Chim.  et  de  Phys.  10  (1819),  p.  29. 

(55)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  15  (1911),  p.  179. 

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Dresden,  1912. 

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(1909),  p.  105. 

(59)  Rohonyi,  H.,  Biochem.  Zeit.  44  (1912),  p.  165. 

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(62)  Schiff,  H.,  Ber.  d.  d.  chem.  Ges.  30  (1897),  p.  2449.    Annalen  der  Chem. 

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543. 

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(1911),  p.  459. 


CHAPTER  II 
THE  PREPARATION   OF  PURE   PROTEINS 

1.  The  Proteins  as  Chemical  Individuals.  —  With  the  excep- 
tion of  casein  and  the  protamins  and  those  proteins  such  as 
haemoglobin,  fibrin,  egg  albumin  and  certain  of  the  vegetable 
proteins  which  may  be  prepared  in  crystalline  condition,  it  can- 
not be  positively  affirmed  that  any  of  the  proteins  which  have 
hitherto  been  isolated  in  a  "pure"  condition  are,  in  reality, 
chemical  individuals  and  not  mixtures,  tolerably  constant  in 
composition,  of  two  or  more  proteins  or  of  one  protein  with  the 
products  of  its  partial  hydrolysis  or  with  other  colloidal  sub- 
stances. Our  methods  of  isolation  and  purification  are  admittedly 
inadequate,  and  the  most  diverse  opinions  exist  regarding  the 
appropriate  criteria  of  the  purity  of  any  given  protein.  The 
reason  for  this  latter  fact  is  probably  to  be  sought  in  the  imper- 
fection of  our  knowledge  of  the  properties,  chemical,  physical, 
and  physico-chemical,  of  the  proteins  as  a  class;  lack  of  knowledge 
implying,  of  course,  the  lack  of  a  basis  for  comparison  and  of  a 
standard  for  calibration. 

The  proteins  are,  as  a  rule,  non-crystallizable  or  only  crystal- 
lizable  with  difficulty  and  under  such  conditions  as  to  involve 
contamination  with  a  variable  and  usually  indeterminate  pro- 
portion of  impurities,  while  repeated  re-crystallization  usually 
leads  to  a  more  or  less  sensible  alteration  in  the  properties  of  the 
protein,  which  may  be  attributable,  in  the  majority  of  cases,  to 
a  partial  hydrolysis.  The  salts  which  the  proteins  form  with  the 
inorganic  bases  and  acids  are,  as  a  rule,  either  soluble  or  else,  if 
insoluble,  of  such  a  nature  (for  example,  the  protein  salts  of  the 
heavy  metals)  that  the  inorganic  constituent  cannot  be  removed 
again  from  the  molecule  without  altering  its  properties. 

A  limited  number  of  the  proteins  are  insoluble  in  distilled  water 
when  uncombined  with  bases  or  acids,  and  these,  of  course, 
afford  exceptionally  favorable  opportunities  for  isolation  and  puri- 
fication. Precipitation  of  these  proteins  in  the  free  condition  can 

34 


PROTEINS  AS  CHEMICAL  INDIVIDUALS  35 

usually  be  brought  about  by  inducing  a  certain  reaction  (i.e., 
hydrogen  or  hydroxyl  ion  concentration)  in  their  solution,  through 
the  addition  of  acids  or  of  bases,  the  hydrogen-  or  hydroxyl-ion 
concentration  being  sufficient  to  set  the  protein  free  from  its 
combination  with  bases  or  acids  respectively  and  insufficient  to 
lead  to  the  formation  of  a  new  salt  with  the  acid  or  base  employed 
for  its  precipitation.  Thus,  for  example,  casein  is  precipitated 
from  milk  by  the  addition  of  acetic  acid,  "insoluble"  serum  globulin 
from  serum  by  dilution  and  the  passage  of  C02,  histones  from  cell- 
extracts  by  the  addition  of  ammonia. 

The  isolation  of  proteins  of  this  class  is  usually  most  conven- 
iently carried  out  by  employing  for  the  precipitation  an  acid  or 
base  which  is  insufficiently  dissociated  to  transform  the  protein 
into  a  base,  if  an  acid  is  employed,  or  into  an  acid  if  a  base  is 
employed  for  the  precipitation,  so  that  the  protein  which  is  at 
first  precipitated  does  not,  on  adding  an  excess  of  the  acid  or  base, 
form  a  salt  and  pass  into  solution  again.  For  instance,  if  a 
" strong"  acid  such  as  hydrochloric  acid  be  added  to  milk,  or  to 
any  solution  of  a  caseinate  of  an  alkali  or  an  alkaline  earth,  free 
casein  is  at  first  precipitated.  But  if  the  addition  of  the  hydro- 
chloric acid  be  continued  the  casein  passes  into  solution  again 
and  it  is  now  found  that  the  casein  is  behaving  as  a  base  and  that 
a  certain  proportion  of  the  acid  is  neutralized  by  it.  If  a  "weak" 
acid  such  as  acetic  be  employed,  however,  the  acidity  of  the  mix- 
ture is  insufficient  to  transform  the  casein  into  a  base,  and  a 
considerable  excess  of  acetic  acid  may  be  added  to  the  mixture 
without  danger  of  loss  of  material  owing  to  resolution  of  the  protein. 

The  acid  or  base  which  is  employed  in  setting  free  the  insoluble 
uncombined  proteins  of  this  class  must,  however,  be  sufficiently 
strong  to  set  free  the  protein  from  its  salts;  for  example,  C02 
cannot  be  employed  to  precipitate  casein  because  casein  is  a 
stronger  acid  than  C02  and  displaces  it  from  carbonates.  But 
C02  can  be  and  is  successfully  employed  to  precipitate  "insoluble" 
serum- -globulin  because  this  protein  is  a  weaker  acid  than  casein 
and  can  be  displaced  from  its  salts  by  so  feeble  an  acid  as  car- 
bonic acid.  Undoubtedly  correlated  with  this  is  the  fact  that 
serum  globulin  is  more  readily  transformed  into  a  base  than 
casein,  so  that  acetic  acid  redissolves  and  is  partially  neutralized 
by  serum  globulin,  although  it  is  insufficiently  strong  except  in 
very  high  concentrations  to  redissolve  casein. 


36  CHEMICAL  STATICS 

It  is  clear,  therefore,  that  proteins  of  this  class  must  be  pre- 
cipitable  only  within  a  very  narrow  range  of  hydrogen-  and 
hydroxyl-ion  concentrations  and  that  the  probability  of  simul- 
taneous precipitation  of  more  than  one  protein  is  very  slight 
unless  the  proteins  chance  to  be  acids  or  bases  of  very  nearly 
equal  strength.  In  very  few  cases,  however,  do  we  possess,  at 
present,  any  very  reliable  guarantee  that  this  does  not  occur, 
and  in  a  few  instances,  at  all  events,  we  are  positive  that  it  does 
occur.  For  example,  the  various  members  of  the  paranuclein 
series,  which  are  derived  from  casein  by  its  partial  hydrolysis, 
are  all  precipitable  by  acetic  acid,  and,  some  of  them  at  least, 
are  not  redissolved  by  the  addition  of  moderate  excess  of  acetic 
acid.  True,  there  are  recognizable  differences  between  the  basic 
functions  of  the  various  paranucleins  and  casein.  The  para- 
nucleins  are  precipitated  in  solutions  of  lower  acidity  than  casein, 
and  doubtless  an  acid  could  be  found  the  solution  of  which,  at 
a  concentration  sufficiently  acid  to  precipitate  paranuclein,  would 
not  be  sufficiently  acid  to  precipitate  casein.  But  this  acid  has 
not,  as  yet,  been  discovered  and  so  a  reliable  method  for  sepa- 
rating casein  from  paranuclein  in  a  solution  containing  a  mixture 
of  these  proteins  is  yet  to  seek;  while  the  separation  of  the  dif- 
ferent members  of  the  paranuclein  group  from  one  another  is  a 
task  replete  with  difficulties  and  uncertainties.* 

It  is  not  intended  to  imply,  by  the  above  remarks,  that  the 
isolation  of  free  casein  is  as  a  rule  attended  with  danger  of  con- 
tamination with  paranuclein,  because,  fortunately,  the  presence 
of  paranuclein  in  solutions  of  casein  is  not  to  be  feared  unless  the 
casein  has  undergone  appreciable  hydrolysis  in  an  acid  medium. 
I  am  merely  pointing  out  the  imaginary  difficulties  which  would 
be  encountered  in  endeavoring  to  separate  casein  from  para- 
nuclein, were  they  found  in  nature  in  the  same  fluid,  as  an  illus- 
tration of  the  very  real  difficulties  which  unquestionably  attend 
the  separation  and  isolation  of  other  proteins  which  are  insoluble 
in  distilled  water  when  uncombined  with  acids  or  bases. 

All  that  can  be  said,  therefore,  when  a  protein  isolated  by  means 
of  a  certain  procedure,  is  found  repeatedly  to  yield  the  same 
physical,  physico-chemical  and  chemical  constants,  is  that  it  is 
a  protein  or  mixture  of  proteins  of  constant  composition  provided 
that  particular  method  of  isolation  be  employed  and  no  other. 
*  Vide  T.  Brailsford  Robertson  (31), 


CASEIN  37 

Only  when  a  protein  has  been  prepared  in  crystalline  form  or 
by  many  different  observers  in  many  different  ways  and  has 
always  been  found  to  possess  the  same  physical,  physico-chemical 
and  chemical  constants,  can  we  pronounce  it,  with  any  degree 
of  safety,  a  definite  chemical  individual.  So  far  this  has  only 
been  accomplished  for  a  very  limited  number  of  proteins. 

It  cannot  with  propriety  be  assumed,  therefore,  that  the  fol- 
lowing methods  for  the  isolation  of  "pure"  proteins  yield  definite 
chemical  individuals,  save  those  for  the  isolation  of  the  crystal- 
line proteins  and  of  casein  and  of  the  various  members  of  the 
protamin  group.  But  the  methods  which  follow  are  those  which, 
to  the  knowledge  of  the  author,  have  been  shown  to  yield,  at 
least,  mixtures  of  appreciably  uniform  composition,  appreciably  free 
from  non-protein  contamination. 

Very  many  well-known  methods  for  the  isolation  and  purifi- 
cation of  various  proteins  are,  of  course,  omitted,  not  because 
they  do  not  in  all  probability  yield  products  conforming  to  the 
above  restrictions,  but  because  either  evidence  of  this  fact  is 
lacking,  or  they  have  never  been  employed  in  research  of  a  dis- 
tinctly physico-chemical  character.* 

2.  Casein.  —  The  following  is  the  method  of  preparation 
employed  by  Hammarsten  (8) : 

"Milk  is  diluted  with  four  volumes  of  water  and  the  mixture 
treated  with  acetic  acid  to  0.75  to  1  per  mille.  Casein  thus  ob- 
tained is  purified  by  repeatedly  dissolving  in  water  with  the  aid 
of  the  smallest  quantity  of  alkali  possible,  by  filtering  and  re- 
precipitating  with  acetic  acid  and  thoroughly  washing  with  water. 
Most  of  the  milk-fat  is  retained  by  the  filter  on  the  first  filtration, 
and  the  casein  contaminated  with  traces  of  fat  is  purified  by 
treating  with  alcohol  and  ether." 

According  to  Bosworth  and  Van  Slyke  (3)  casein  prepared  in 
this  manner  is  always  contaminated  by  a  small  admixture  of 
dicalcium  phosphate  and  contains  about  0.14  per  cent  of  phos- 
phorus attributable  to  this  source.  The  method  of  preparation 
which  these  investigators  employ  to  secure  a  product  free  from 
this  contamination  is  as  follows: 

Separator  skim-milk  is  diluted  with  seven  or  eight  times  its 
volume  of  distilled  water,  and  dilute  acetic  acid  (6  cc.  of  glacial 

*  For  a  number  of  methods  for  the  separation  of  various  animal  proteins 
vide  Hammarsten  (8). 


38  CHEMICAL  STATICS 

acetic  acid  diluted  to  1  litre)  is  carefully  added  until  the  casein 
separates  completely,  after  which  the  clear  solution  is  removed 
by  syphon  as  soon  as  the  precipitate  settles.  Distilled  water  is 
then  added,  the  mixture  stirred  vigorously  and  the  precipitate 
allowed  to  settle,  after  which  the  wash  water  is  syphoned  off. 
Water  is  again  added,  and  the  casein  is  dissolved  by  adding,  for 
each  litre  of  milk  used,  1  litre  of  dilute  NH4OH  (6  cc.  of  strong 
reagent  diluted  to  1  litre).  When  the  solution  is  complete  the 
whole  is  filtered  through  a  thick  layer  of  absorbent  cotton.  The 
casein  is  then  precipitated  again  with  dilute  acetic  acid;  the 
precipitate  is  allowed  to  settle,  and  is  then  washed,  redissolved 
in  dilute  NH4OH  and  filtered,  the  process  of  precipitation,  wash- 
ing, dissolving,  etc.,  being  repeated  not  less  than  four  times. 
Finally  an  excess  of  strong  NH4OH  (10  cc.)  is  added  and  then 
20  cc.  of  a  saturated  solution  of  ammonium  oxalate.  The  mix- 
ture is  allowed  to  stand  twelve  hours  or  more.  Calcium  is  pre- 
cipitated as  oxalate  in  very  finely  divided  condition,  too  fine  to 
permit  its  satisfactory  removal  by  ordinary  methods  of  filtration. 
Better  aggregation  of  the  precipitate  can,  however,  be  effected 
by  means  of  centrifugal  force.  The  centrifuged  mixture  is  then 
filtered  through  double  thickness  of  filter  paper.  The  filtered 
solution  is  next  treated  with  dilute  HC1  (10  cc.  HC1,  sp.  gr.  1.20 
diluted  to  1  litre)  until  the  casein  is  precipitated.  The  precipi- 
tate is  washed  with  distilled  water  until  free  from  chloride  and  is 
then  placed  on  a  hardened  filter  paper  in  a  Buchner  funnel,  as 
much  water  as  possible  being  now  removed  by  suction.  The  mass 
is  next  transferred  to  a  large  mortar  and  thoroughly  triturated 
with  95  per  cent  alcohol.  The  alcohol  is  then  removed  by  suc- 
tion on  a  Buchner  funnel  and  the  casein  is  placed  in  a  mortar 
and  triturated  with  absolute  alcohol.  Most  of  the  alcohol  is 
removed  and  the  casein  treated  twice  with  ether  in  a  mortar 
by  trituration,  the  ether  being  removed  each  time  by  means  of 
suction  on  a  Buchner  funnel.  The  material  is  then  placed  in  a 
large  evaporating  dish  and  spread  out  in  a  layer  as  thin  as  possible, 
allowed  to  stand  twelve  hours  or  more  in  a  warm  place,  and 
then  finely  ground  in  a  mortar  until  the  particles  pass  through  a 
40-mesh  sieve  and  dried  for  two  days  over  H2S04  in  a  desiccator 
under  diminished  pressure. 

Casein  prepared  in  this  manner  contains  only  0.71  per  cent  of 
phosphorus;  corresponding  almost  exactly  to  the  theoretical  per- 


CASEIN  39 

centage  calculated  on  the  supposition  that  casein  contains  two 
atoms  each  of  phosphorus  and  sulphur.  Previous  estimates, 
derived  from  analyses  of  casein  prepared  by  Hammarsten's 
method,  indicated  a  phosphorus  content  of  about  0.85  per 
cent. 

Casein  "nach  Hammarsten"  is  obtainable  upon  the  market  in 
a  high  degree  of  purity.  For  the  investigations  described  in  this 
work  I  have  always  employed  the  C.  P.  casein  "nach  Hammar- 
sten" which  is  manufactured  in  Germany  for  Messrs.  Eimer  and 
Amend  of  New  York.  This  substance  is  free  from  appreciable 
fat  and  would  appear  to  be  contaminated  only  by  a  small  amount 
of  an  acid,  water-soluble  substance.  This  I  remove  by  further 
purification  in  the  following  manner  (30)  (35) : 

Half  a  pound  of  casein  is  triturated  with  about  12  litres  of  dis- 
tilled water,  the  water  being  added  in  six  successive  portions. 
On  each  addition  of  water  the  casein  is  well  stirred  up  in  a 
porcelain  mortar  and  allowed  to  settle,  the  supernatant  water 
is  then  poured  off  and  fresh  water  is  added.  It  is  next  washed 
in  a  similar  manner  in  5  kilos  of  absolute  alcohol,  and  then  in 
5  kilos  of  ether  "  distilled  over  sodium."  The  mortar,  containing 
the  casein  drained  as  free  from  superfluous  ether  as  possible,*  is 
then  placed  in  an  incubator  over  sulphuric  acid  at  40°-50°  C., 
the  flame  is  turned  out  under  the  incubator,  and  it  is  allowed  to 
cool  for  about  24  hours.  The  casein  is  now  found,  if  these  op- 
erations have  been  conducted  carefully,  to  be  in  the  form  of  a 
dry,  pure  white  powder,  still  containing,  however,  a  considerable 
quantity  of  ether.  The  presence  of  a  color  in  the  powder  indi- 
cates contamination,  either  with  moisture  or  with  some  other 
impurity. 

The  casein  is  now  spread  out,  within  the  incubator,  in  a  layer 
not  over  1  cm.  deep,  the  flame  under  the  incubator  is  lighted  and 
it  is  allowed  to  stand  for  24  hours  at  40°-50°  C.  The  casein  is 
then  found  to  be  free  from  appreciable  water  or  ether. 

The  casein  which  is  thus  prepared  gives  every  indication  of  being 
a  pure  product  and  a  definite  chemical  individual.  It  is  insoluble 
in  distilled  water,  save  in  traces  which  adhere  to  the  casein  par- 

*  At  this  point  it  is  necessary  to  avoid  exposing  the  mortar  to  the  moist  air 
of  the  room  for  a  moment  longer  than  is  necessary,  otherwise  the  evaporating 
ether  causes  the  condensation  of  sufficient  moisture  to  spoil  the  product  unless 
it  is  again  treated  with  alcohol  and  ether. 


40  CHEMICAL  STATICS 

tides,*  and  it  neutralizes,  to  phenolphthalein  and  to  litmus,  exactly 
the  quantities  of  alkali  determined  by  Soldner  (41),  Lacquer  and 
Sackur  (15)  and  Van  Slyke  and  Hart  (45). 

Casein  which  has  been  carefully  prepared  in  the  manner  out- 
lined above,  floats  upon  the  top  of  and  is  not  readily  wetted  by 
water  or  watery  solutions  of  bases;  if,  however,  it  contains  a 
mere  trace  of  moisture,  it  is  readily  wetted  by  all  save  the  most 
alkaline  solutions.  In  order  to  successfully  and  completely  dis- 
solve perfectly  anhydrous  casein  it  is  necessary  to  first  add  to  it 
a  very  little  of  the  solution  in  which  it  is  to  be  dissolved,  rub  it 
up  into  a  paste,  and  then  add,  while  stirring,  the  remainder  of 
the  solution. 

If  casein  which  is  readily  wetted  by  water  be  desired  (34)  it  is 
necessary  to  omit  the  desiccation  over  H2SO4.  For  this  purpose 
CaCl2  should  be  employed  as  the  desiccating  agent,  and  desic- 
cation should  be  continued  for  12  hours  at  about  30°  C.  Casein 
prepared  in  this  way  readily  sinks  in  and  is  wetted  by  water  and 
aqueous  solutions;  it  contains,  however,  not  very  significant  traces 
of  moisture  and  a  considerable  amount  of  adherent  ether. 

3.  *  Insoluble"  Serum  Globulin.  —  There  appear  to  be  at 
least  two  globulins  in  serum,  the  one  insoluble  in  distilled  water, 
which  is  precipitated  on  dialysis  of  the  serum,  or  dilution  and  the 
subsequent  addition  of  a  weak  acid;  the  other  soluble  in  water, 
precipitable  by  saturation  with  magnesium  sulphate  after  the 
removal  of  the  insoluble  fraction  (16)  (5)  (27). 

According  to  Hardy  these  globulins  differ  markedly  in  their 
phosphorus  content,  the  insoluble  globulin  containing  from  0.07 
to  0.08  per  cent  phosphorus;  the  soluble  globulin  only  a  trace 
(about  0.009  per  cent)  of  phosphorus  (9). 

According  to  Hammarsten  (7)  the  individuality  of  these  pro- 
teins, and  the  freedom  of  any  given  preparation  of  the  one  from 
an  admixture  of  the  other  is  open  to  serious  doubt.  Moreover, 
as  Taylor  has  shown  (44),  the  "insoluble"  globulin  is  readily 
converted,  by  hydrolysis,  into  the  "soluble"  form.  The  relation- 
ship between  the  various  serum  globulins  would  therefore  appear 
to  be  somewhat  analogous  to  that  between  the  various  members 
of  the  paranuclein  group.  . 

*  So  that  when  litmus  paper  is  dipped  into  a  suspension  of  casein  particles 
in  distilled  water  the  paper  is  reddened  where  it  is  touched  by  the  undissolved 
particles,  while  the  fluid  which  bathes  them  remains  clear  and  neutral  (30). 


SERUM   GLOBULIN  41 

The  following  method  of  preparing  serum  globulin  is  based 
upon  the  observation  of  Alexander  Schmidt  (38)  and  Kuhne  (14) 
that  " insoluble"  serum  globulin  can  be  precipitated  from  serum, 
previously  diluted  with  ten  volumes  of  water,  by  the  passage  of 
C02.  It  appears  to  be  difficult  or  impossible  to  bring  about  this 
precipitation  in  the  total  absence  of  salts  of  stronger  acids,  e.g., 
NaCl.  I  have  repeatedly  observed  that  if  serum  globulin,  pre- 
cipitated by  C02  and  carefully  washed,  be  dissolved  in  a  minimal 
quantity  of  NaOH  or  KOH  it  cannot  be  reprecipitated  by  dilu- 
tion and  the  passage  of  C02,  although  a  distinctly  acid  and  very 
opaque  solution  results. 

Three  litres  of  ox-serum  are  diluted  with  ten  volumes  of  dis- 
tilled water,  and  C02  is  bubbled  through  it  at  a  good  rate  for 
about  half  an  hour.  The  globulin  which  is  thus  precipitated 
is  allowed  to  settle  in  tall  glass  cylinders,*  the  supernatant 
fluid  being  syphoned  off  after  settling.  The  precipitate  is  then 
washed  with  about  60  litres  of  distilled  water,  in  two  portions. 
The  globulin  is  then  dissolved  in  a  minimal  quantity  of 
N / 10  HClf  and  immediately  reprecipitated  by  cautious  neutral- 
ization with  AT/10  KOH.  This  precipitate,  after  settling  and  de- 
cantation  of  the  supernatant  fluid,  is  washed  in  60  litres  of  distilled 

*  For  the  settling  and  washing  of  protein  precipitates  I  employ  wide- 
mouthed  glass  cylinders  from  50  to  60  cm.  high  and  about  10  cm.  in  diameter, 
closed  by  ground  glass  stoppers.  The  syphon  is  provided  with  a  side  tube 
through  which  it  can  be  filled  and  can  then  be  closed  by  a  rubber  tube  provided 
with  a  pinch-cock.  When  the  protein  is  being  washed  with  alcohol  or  with 
ether,  the  syphon  is  filled  with  alcohol  so  as  to  avoid  accidental  contamination 
of  the  contents  of  the  cylinder  with  water.  The  syphon  is  supported  on  a 
stand  which  is  so  arranged  that  the  height  of  the  opening  of  the  syphon  above 
the  precipitate  can  be  adjusted  by  turning  a  screw.  The  greater  part  of  the 
liquid  is  first  rapidly  run  off  with  the  opening  of  the  syphon  a  good  way  above 
the  precipitate.  The  syphon  is  then  lowered  and  the  remainder  of  the  liquid 
run  off  more  slowly. 

f  This  can  be  calculated  from  the  fact  that  1  litre  of  serum  yields  about 
5  grams  of  globulin  and  that  about  20  X  10~6  gram  equivalents  of  the  strong 
monobasic  acids  just  suffice  to  dissolve  one  gram.  If  acetic  acid  be  preferred, 
about  100  X  10~5  equivalents  are  required  to  dissolve  1  gram  (9). 

It  has  been  shown  by  Hammarsten  (7)  that  "insoluble"  serum  globulin 
is  not  readily  "denatured,"  i.e.,  altered  in  properties,  by  acids.  "A  solution 
of  serum  globulin  containing  0.2-0.3  per  cent  acetic  acid  remains  unchanged 
for  days  at  a  low  temperature,  and  one  can  recover  the  serum  globulin  unal- 
tered by  neutralization."  In  the  presence  of  stronger  acids  denaturation  is  a 
matter  of  some  hours. 


42  CHEMICAL  STATICS 

water  in  6  successive  washings,  the  precipitate,  after  each  agi- 
tation with  distilled  water,  being  allowed  to  settle  for  24  hours 
in  the  presence  of  toluol,  after  which  the  supernatant  fluid  is 
drawn  off  and  the  globulin  suspended  in  a  fresh  quantum  of  dis- 
tilled water.*  The  thick  suspension  of  globulin  which  is  thus 
obtained  after  the  final  washing  is  kept,  in  the  presence  of  toluol, 
in  a  stoppered  bottle  and  used  in  this  form,  since  globulin,  if 
washed  with  alcohol  and  ether  and  dried,  is  redissolved  only  with 
difficulty. 

The  suspension  is  well  shaken  before  withdrawing  a  measured 
sample.  The  globulin  content  of  the  suspension  is  determined  to 
within  ±0.01  gram  per  100  cc.  by  placing  25  cc.  samples  in  small 
and  accurately  weighed  beakers,  evaporating  the  fluid  to  dry- 
ness  on  a  water-bath,  and  then  drying  the  residue  over  H2S04  at 
70°  until  its  weight  becomes  constant. 

4.  Fibrin.  —  The  following  is  the  method  of  preparing  fibrin 
which  is  employed  by  Bosworth  (2). 

Fresh  ox-blood  is  collected  in  a  large  bottle  and,  as  soon  as 
possible,  transferred  to  wide-mouthed  precipitating  jars  and 
allowed  to  coagulate.  The  clots  are  then  removed,  broken  into 
small  pieces,  and  washed  in  running  water  to  remove  the  serum 
and  blood  corpuscles.  The  washed  masses  of  fibrin  are  passed 
through  a  mincing  machine,  placed  in  a  large  (8-litre)  bottle,  a 
little  toluol  added,  and  the  bottle  filled  with  0.2  per  cent  sodium 
hydroxide  solution.  This  solution  causes  the  fibrin  to  swell  and 
after  about  36  hours  the  whole  content  of  the  bottle  resembles  a 
thin  jelly.  This  jelly  is  broken  up,  one-half  transferred  to  another 
8-litre  bottle,  and  after  the  two  bottles  are  filled  by  the  addition 
of  water  they  are  allowed  to  stand  for  an  additional  36  hours. 
The  jelly  is  by  then  almost  completely  dissolved  and  the  contents 
of  both  bottles  are  filtered,  first  through  cheese-cloth,  then  linen 
and  finally  paper.  The  clear  filtrate  is  then  diluted  with  an  equal 
volume  of  water  and  placed  in  tall  wide-mouthed  precipitating 
jars.  Dilute  acetic  acid  (0.3  per  cent)  is  then  added  cautiously. 
At  a  certain  point  a  flocculent  precipitate  of  fibrin  appears  which 
quickly  settles  to  the  bottom  of  the  jars. 

*  This  suspension  is,  of  course,  equivalent  to  a  prolonged  dialysis  of  the 
protein,  the  precipitate  acting  as  a  dialyser  of  enormously  extended  surface, 
permitting  the  passage  of  associated  diffusible  impurities  into  the  distilled 
water  and  retaining  the  protein. 


HAEMOGLOBIN  43 

The  supernatant  liquid  is  syphoned  off,  the  precipitate  washed 
with  water,  dissolved  in  dilute  sodium  hydroxide  (0.05  per  cent) 
and  again  precipitated  with  acetic  acid.  This  process  is  repeated 
three  times,  the  final  precipitate  being  washed  with  alcohol  and 
ether  and  dried  over  sulphuric  acid  in  an  evacuated  desiccator. 

The  behavior  of  fibrin  strongly  resembles  that  of  casein  in 
all  stages  of  its  preparation  except  in  its  extreme  sensitiveness 
to  a  slight  excess  of  acid  or  alkali,  for  unlike  casein,  it  is  readily 
soluble  in  weak  acetic  acid. 

5.  Haemoglobin.  —  The  following  is  the  method  of  prepara- 
tion recommended  by  Preyer  (26). 

The  blood  is  collected  in  a  vessel  and  allowed  to  coagulate 
and  to  stand  for  several  hours  (or,  better,  for  a  day)  in  a  cool 
place.  Then  the  serum  with  the  white  corpuscles  and  the  fat 
which  have  collected  above  the  clot  is  removed  and  the  coag- 
ulum  is  washed  with  distilled  water  and  then  cut  into  very  small 
pieces,  and  these  pieces  in  turn  are  repeatedly  washed  with  cold 
distilled  water.  Then  the  clot  is  comminuted,  best  by  freezing 
and  reducing  the  frozen  mass  to  powder.  This  powder  is  placed 
in  a  filter  paper  and  washed  with  cold  distilled  water  until  the 
filtrate  no  longer  gives  any  very  bulky  precipitate  with  bichloride 
of  mercury.  The  coagulum  is  extracted  by  water  heated  to  from 
30  to  40  degrees  and  filtered,  and  the  filtrate  is  collected  in  a 
large  cylindrical  vessel  standing  in  ice.  A  small  measured  portion  of 
the  red  solution  thus  obtained  is  gradually  mixed  during  constant 
agitation  with  small  quantities  of  alcohol  until  a  slight  precipi- 
tate forms.  This  determines  how  much  alcohol  may  be  added  to 
the  whole  solution  without  a  precipitate  appearing.  A  slightly 
smaller  proportion  of  alcohol  is  now  added  to  the  remaining 
filtrate  and  the  mixture  is  placed  in  a  cooling,  medium.  After  a 
few  hours  the  crystals  separate  in  great  abundance.  The  crys- 
tals, owing  to  the  large  volume  of  water  used,  are  very  easily 
filtered  off  in  the  cold.  They  are  then  washed  with  cold  water 
containing  a  little  alcohol  until  the  filtrate  yields  only  an  insig- 
nificant cloudiness  upon  the  addition  of  acetate  of  lead  or  mer- 
curic chloride.  The  product  yielded  is  a  very  large  one.  The 
crystals  may  be  purified  by  repeated  washing  by  decantation 
until  the  wash-water  does  not  become  cloudy  with  bichloride  of 
mercury,  lead  acetate  or  silver  nitrate.  They  are  then  nearly 
pure,  and  the  ash  is  free  from  phosphoric  acid  and  consists  of 


44  CHEMICAL  STATICS 

pure  iron  oxide.  If  this  is  not  the  case,  then  they  must  be  dis- 
solved in  warm  water  and  recrystallized.  At  a  temperature  of 
less  than  0°  C.  the  crystals  can  be  dried  in  the  air  without  de- 
composition. 

Preyer  states  that,  of  all  kinds  of  blood,  that  of  the  horse  is 
best  adapted  for  the  production  of  very  large  quantities  of  pure 
haemoglobin. 

For  a  variety  of  rapid  methods  of  preparing  small  quantities 
of  crystals  from  the  blood  of  different  species  the  reader  is  re- 
ferred to  the  monograph  on  the  Crystallography  of  Haemoglobins 
by  Reichert  and  Brown  (28). 

6.  Crystallizable  Egg- Albumin.  —  The  method  of  Hopkins  and 
Pinkus  ( 1 0)  is  universally  employed  for  the  preparation  of  this  protein . 

Two  hundred  cc.  of  egg-white  obtained  from  newly-laid  fowls' 
eggs  are  mixed  with  an  equal  bulk  of  saturated  ammonium  sul- 
phate solution,  the  latter  being  very  gradually  added  and  the  mix- 
ture stirred  briskly  with  an  egg-beater  between  each  addition. 
It  is  then  allowed  to  stand  over  night.  The  mixture  is  then  filtered 
and  to  the  clear  filtrate  more  saturated  ammonium  sulphate  solu- 
tion is  added  until  a  permanent  precipitate  is  obtained.  Distilled 
water  is  then  added,  a  few  drops  at  a  time,  until  the  solution  is 
just  clear  again.  Ten  per  cent  acetic  acid  solution  is  then  added 
drop  by  drop  until  a  slight  but  definite  precipitate  has  appeared. 
The  bottle  is  then  immediately  stoppered  and  allowed  to  stand. 
After  24  hours  an  abundance  of  uniformly  crystalline  precipitate 
has  settled  out  consisting  of  rosettes  of  needles.  If  a  somewhat 
greater  proportion  of  acetic  acid  is  employed  the  rosettes  are 
mixed  with  sheaths  and  fan-shaped  aggregates  of  crystals.  Re- 
crystallization  of  this  first  product  is  carried  out  by  filtering  off 
the  crystals  from  the  mother-liquor,  redissolving  in  a  moderate 
amount  of  water,  acidifying  with  a  few  drops  of  dilute  acetic 
acid  and  adding  saturated  ammonium  sulphate  solution  until  a 
faint  turbidity  is  produced.  In  24  hours  a  large  proportion  of 
the  protein  will  have  recrystallized.  This  process  may  be  re- 
peated 5  or  6  times  in  the  course  of  a  week.  The  crystals  which 
are  finally  obtained  may  be  freed  from  inorganic  contamination 
by  prolonged  dialysis  against  running  distilled  water. 

7.  Ovovitellin.  —  The  following  method  of  preparation  is  that 
employed  by  Osborne  and  Campbell  (23),  modified  by  Plimmer 
(25)  and  further  modified  by  the  author  (33). 


OVOVITELLIN  45 

Twenty-five  yolks  of  eggs  are  carefully  washed,  without  break- 
ing the  enveloping  membrane,  in  a  stream  of  water,  so  as  to  re- 
move all  traces  of  the  whites.  To  the  yolk  is  then  added  an  equal 
volume  of  10  per  cent  sodium  chloride  solution  and  the  solution 
which  is  thus  obtained  is  extracted  from  ten  to  twelve  times 
with  twice  its  volume  of  ether  in  separatory  funnels,  extracting 
several  times  after  the  ethereal  layers  fail  to  yield  a  precipitate, 
due  to  the  presence  of  lecithin,  upon  the  addition  of  acetone. 
The  complete  extraction  occupies  a  period  of  from  two  to  three 
weeks.  The  watery  layer  which  is  finally  obtained  is  then  poured 
into  twenty  volumes  of  distilled  water,  and  the  precipitate  of 
ovovitellin  which  is  thus  obtained  is  allowed  to  settle  in  tall 
glass  cylinders.  The  supernatant  fluid  is  then  syphoned  off  and 
the  precipitate  redissolved  in  10  per  cent  sodium  chloride  and 
reprecipitated  in  the  same  manner.  This  process  is  repeated. 
Finally  the  vitellin  is  dissolved  in  very  dilute  sodium  hydrate 
and  the  solution  filtered,  the  filtrate  being  allowed  to  drop  directly 
into  dilute  acetic  acid,  thus  reprecipitating  the  vitellin.  This 
precipitate  is  suspended  in  distilled  water  and  allowed  to  settle 
in  tall  glass  cylinders.  The  supernatant  water  is  then  drawn  off 
and  the  washing  with  water  repeated  several  times.  The  pre- 
cipitate is  then  washed  in  6  litres  of  99.8  per  cent  alcohol.  After 
allowing  the  precipitate  to  settle,  the  supernatant  alcohol  is 
syphoned  off  and  the  washing  in  alcohol  repeated  twice.  The 
vitellin  is  then  washed  twice  in  ether  "distilled  over  sodium," 
employing  6  litres  each  time.  The  thick  suspension  of  vitellin 
in  ether  finally  obtained  is  quickly  poured  into  a  hardened  filter 
and  allowed  to  filter  and  dry  over  sulphuric  acid  at  40  degrees 
for  48  hours.  The  vitellin  is  thus  obtained  as  a  white,  somewhat 
coarse  powder. 

About  a  gram  of  this  ovovitellin  was  placed  in  about  30  cc. 
of  alcohol  and  boiled  for  some  five  minutes.  About  5  cc.  of  the 
alcohol,  after  filtration,  was  then  tested  directly  for  lecithin  by 
the  addition  of  several  volumes  of  acetone.  The  remainder  of 
the  alcohol  was  evaporated  down  to  dryness  and  the  residue 
(barely  visible)  taken  up  in  about  10  cc.  of  ether.  This  ether 
was  then  tested  by  the  addition  of  several  volumes  of  acetone. 
Both  tests  proved  entirely  negative,  not  the  slightest  opalescence 
being  produced  by  the  acetone;  thus  the  ovovitellin  did  not  con- 
tain any  lecithin.  Hence  the  older  statements  to  the  effect  that 


46  CHEMICAL  STATICS 

vitellin  is  always  associated  and  probably  combined  with  lecithin 
are  attributable  to  inadequate  extraction  of  the  yolks  with  ether 
and  the  consequent  incomplete  removal  of  the  lecithin. 

It  is  extremely  difficult  to  prepare  homogeneous  solutions  of 
ovo vitellin.  A  five  per  cent  solution  in  alkali  (JV/10  KOH)  is  a 
thick  jelly,  opalescent  owing  to  the  presence  of  a  multitude  of 
air  bubbles  entangled  in  it  while  stirring.  In  endeavoring  to 
prepare  more  dilute  solutions  it  is  found  very  difficult  to  avoid 
the  formation  of  small  lumps  of  jelly  within  the  solutions,  and 
these  are  exceedingly  difficult  to  break  up,  and  dissolve  with  ex- 
treme slowness.  Although  vitellin  is  soluble  in  dilute  solutions 
of  the  strong  acids,  yet  when  the  powder  is  directly  mixed  with 
an  acid  solution  it  will  not  dissolve  or  does  so  with  extreme 
slowness;  it  is  soluble  in  acid  solutions  only  when  freshly  pre- 
cipitated and  still  wet.* 

It  is  possible  to  obtain  clear,  homogeneous  1  per  cent  solutions 
of  ovovitellin  (not  more  concentrated)  by  gradually  dropping 
the  vitellin  from  above  into  the  solvent  while  undergoing  violent 
stirring,  and  maintaining  the  stirring  for  about  an  hour. 

8.  The  Vegetable  Proteins.  —  The  following  methods  of 
preparing  the  globulin  of  flax-seed  will  serve  as  an  illustration  of 
the  general  nature  of  the  technique  which  is  employed  in  pre- 
paring the  various  vegetable  proteins.  For  further  information 
concerning  this  subject  the  reader  is  referred  to  the  monograph 
by  Osborne  (22)  in  which  he  summarizes  the  result  of  his  impor- 
tant and  extensive  researches  upon  the  preparation  and  properties 
of  the  vegetable  proteins.  According  to  Osborne  (20)  flax-seed 
globulin  may  be  extracted  from  flax-seed  meal  in  either  of  three 
ways,  namely  by  extraction  with  distilled  water,  or  with  sodium 
chloride  solution  or  with  dilute  KOH.  The  following  are  the 
methods  of  preparation  employed  by  him. 

(a)  Extraction  with  distilled  water. 

The  ground  flax-seed  is  freed  from  oil  by  extracting  with  ben- 
zene and  from  the  greater  part  of  the  outer  coating  of  the  seed 
by  sifting  through  a  fine  sieve.  One  hundred  grams  of  this 
meal  are  extracted  for  about  24  hours  with  distilled  water  at 
temperatures  between  20  and  40°  C.  The  filtered  extract  has 
a  yellow  color,  is  acid  in  reaction  and  very  slightly  turbid. 

*  Exactly  the  same  phenomenon  is  encountered  with  casein.  Cf.  Van 
Slyke  and  Van  Slyke  (46),  T.  Brailsford  Robertson  (32). 


VEGETABLE  PROTEINS  47 

This  extract  is  then  saturated  with  pure  ammonium  sulphate. 
The  copious  precipitate  is  collected  on  a  filter  and,  after  the  com- 
pletion of  filtration,  dissolved  in  about  800  cc.  of  water.  The 
solution  is  then  filtered  and  placed  in  a  dialyser.  After  twenty- 
four  hours  a  considerable  precipitate  has  formed,  which  is  seen 
under  the  microscope  to  consist  entirely  of  perfectly  formed 
octahedral  crystals.  After  dialysing  for  about  five  days  the  solu- 
tion is  found  to  be  free  from  chlorides,  and  the  precipitate  is  then 
filtered  off  and  washed  with  water,  followed  by  dilute  alcohol 
gradually  increased  in  strength  to  absolute  alcohol;  it  is  finally 
washed  with  ether  and  dried  over  sulphuric  acid. 

The  preparation,  after  drying,  still  retains  its  crystalline  char- 
acter. The  meal  yields  about  10  per  cent  of  its  weight  in  globulin. 

(6)  Extraction  with  sodium  chloride  solution. 

One  hundred  grams  of  the  flax-seed  meal  are  extracted  three 
successive  times  with  20  per  cent  sodium  chloride  solution,  and 
the  clear,  filtered,  bright  yellow  extracts  are  united  and  dissolved 
in  10  per  cent  sodium  chloride,  and  this  solution,  after  filtering 
off  an  insoluble  residue,  is  submitted  to  dialysis.  After  five  days 
the  solution,  free  from  chlorides,  is  filtered  and  the  precipitate 
washed  with  water,  alcohol  and  ether  and  dried.  The  prepara- 
tion, after  drying,  still  retains  its  crystalline  character.  One 
hundred  grams  of  meal  yield  about  11.6  grams  of  protein.  This 
protein  is  identical  with  the  globulin  obtained  by  extraction  with 
water. 

(c)  Extraction  with  dilute  KOH  solution. 

Flax-seed  globulin,  prepared  by  either  of  the  above  methods,  is 
soluble  in  dilute  KOH  and  can  be  precipitated  from  this  solution 
unchanged  in  composition  or  properties,  by  cautious  neutraliza- 
tion with  hydrochloric  acid. 

Flax-seed  meal  is  extracted  with  100  cc.  of  0.01  KOH  per 
gram.  After  filtration  it  is  saturated  with  ammonium  sulphate 
and  the  precipitate  filtered  off  and  dissolved  as  completely  as 
possible  in  10  per  cent  sodium  chloride.  This  solution  is  dialysed 
until  the  chlorides  are  removed  and  the  subsequent  treatment  is 
the  same  as  that  employed  in  the  two  previous  preparations. 

This  preparation  is  imperfectly  crystalline,  but  identical  in 
composition  with  the  other  preparations.  One  hundred  grams 
of  meal  yield  about  17.5  grams  of  protein. 

The  plant  globulins  are,  like  serum  globulin,  precipitable  from 


48  CHEMICAL  STATICS 

their  solutions  in  sodium  chloride  by  dilution  and  the  passage  of 
C02.  It  is  stated  by  Osborne,  however,  that  the  separation  which 
is  secured  in  this  manner  is  rarely  so  complete  as  that  which  is 
accomplished  by  dialysis  (22).  The  plant  globulins  are,  accord- 
ing to  Osborne,  very  readily  "denatured"  by  strong  acids,  even 
when  diluted. 

For  the  preparation  of  some  of  the  seed-proteins  it  is  sufficient 
to  extract  the  crushed  seeds  with  hot  sodium  chloride  solution; 
on  cooling,  the  protein  crystallizes  out  spontaneously  (6)  (19) 
(29). 

Osborne  inclines  to  the  view  that  the  majority  of  the  vegetable 
globulins,  instead  of  being  predominantly  acid  substances,  as  they 
are  generally  believed  to  be,  are,  in  reality,  predominantly  basic. 
This  question  will  be  commented  upon  in  a  later  chapter  (Chap.  V). 

9.  The  Alcohol  Soluble  Vegetable  Proteins  (Gliadin,  Zein, 
etc.).  —  The  following  method  of  preparing  gliadin,  based  upon 
that  employed  by  Osborne  and  Harris  (24),  has  been  communi- 
cated to  me  by  Dr.  J.  E.  Greaves: 

Gluten  is  prepared  by  kneading  dough,  made  from  wheat  flour, 
in  a  stream  of  cold  water  until  all  of  the  starch  has  been  washed 
out;  it  is  then  partially  dried  and  a  moisture  determination  is 
made.  The  moist  gluten  thus  obtained  is  finely  chopped  and 
mixed  with  twenty  times  its  weight  of  alcohol  of  such  a  strength 
that  with  the  water  in  the  gluten  it  forms  an  alcoholic  solution 
containing  70  per  cent  of  alcohol  by  volume.  The  mixture  of  gluten 
and  70  per  cent  alcohol  is  then  allowed  to  stand,  with  frequent 
shaking,  for  48  hours.  After  allowing  the  mixture  to  stand  for 
10  hours  the  supernatant  alcoholic  solution  of  gliadin  is  syphoned 
off  and  filtered  through  very  finely  shredded  and  well-washed  and 
dried  asbestos  until  a  perfectly  clear  filtrate  is  obtained.  The  fil- 
trate is  then  evaporated,  under  a  pressure  of  about  \  atmosphere 
until  frothing  prevents  further  concentration.  It  is  then  cooled  and 
very  slowly  poured,  with  constant  stirring,  into  about  one  hundred 
times  its  volume  of  ice-cold  distilled  water,  containing  10  grams 
of  sodium  chloride  per  litre.  The  gummy  mass,  which  usually 
collects  on  the  stirring-rod,  is  dissolved  by  the  addition  of  the 
least  possible  amount  of-  absolute  alcohol,  and  this  solution  is 
evaporated,  under  reduced  pressure,  to  a  thick  syrup;  the  syrup 
is  then  cooled  and  poured,  in  a  very  fine  stream,  with  constant 
stirring,  into  hot  absolute  alcohol.  The  precipitate  is  dissolved 


OVOMUCOID  49 

in  70  per  cent  alcohol  and  this  solution  is  evaporated  under  re- 
duced pressure,  with  the  occasional  addition  of  absolute  alcohol, 
until  a  thick  syrup  is  again  obtained.  The  gliadin  is  precipitated 
from  this  syrup  by  the  method  just  described,  washed  three  times 
with  ether  (distilled  over  sodium),  partially  dried  over  sulphuric 
acid,  ground  up  as  finely  as  possible,  and  then  completely  dried 
over  sulphuric  acid,  in  partial  vacuum,  at  room  temperature. 
The  conditions  of  the  precipitation,  of  course,  involve  contami- 
nation with  NaCl.  According  to  Osborne  (21)  there  is  only  one 
alcohol-soluble  protein  in  flour;  on  standing  in  alcoholic  solution, 
however,  a  protein  insoluble  in  alcohol  or  alcohol-water  mixtures 
is  precipitated  within  a  few  hours.  Kosutany  (13)  believes  that 
this  substance  is  derived  from  gliadin  by  the  splitting  off  of  water 
and  is  identical  with  glutenin,  the  alcohol-insoluble  and  water- 
insoluble  protein  constituent  of  gluten.  According  to  Osborne 
(21),  however,  glutenin  differs  qualitatively  from  gliadin  in  that, 
upon  hydrolysis  with  hydrochloric  acid,  glutenin  yields  glycocoll 
and  lysin,  while  gliadin  yields  neither  of  these  amino-acids. 

10.  Ovomucoid.  —  The  following  method  of  preparing  ovo- 
mucoid  is  a  modification  of  that  originally  employed  by  Morner 
(17)  (33)  (36). 

The  whites  of  eggs  are  beaten  up  to  a  froth  and  allowed  to 
stand  in  shallow  vessels  over  night.  The  subnatant  fluid  is  then 
poured  off,  the  froth  being  rejected.  This  fluid  is  diluted  to  five 
times  its  volume  with  distilled  water,  and  to  every  litre  of  the 
diluted  fluid  is  added  130  cc.  of  approximately  N/IQ  acetic  acid 
(made  up  by  diluting  10  cc.  of  glacial  acetic  acid  to  1750  cc.). 
This  mixture  is  heated  slowly  to  boiling  point,  being  rapidly 
and  uniformly  stirred  meanwhile,  and  after  being  allowed  to  boil 
for  about  3  to  5  minutes,  is  put  aside  in  rather  shallow  vessels 
for  about  12  hours.  At  the  end  of  this  time  most  of  the  coagulum 
has  floated  to  the  top  and  the  clear  pale  yellow  subnatant  fluid  is 
filtered  through  hardened  filter  paper.  Filtration  is  very  rapid, 
and  the  filtered  fluid,  when  boiled,  either  with  or  without  further 
addition  of  acetic  acid,  remains  perfectly  clear.  The  fluid  which 
is  thus  obtained  is  now  slowly  evaporated  to  one-fifth  of  its  volume, 
the  temperature  of  the  fluid  never  being  allowed  to  rise  above 
55°  C.  After  allowing  this  fluid  to  cool,  the  protein  is  precipitated 
from  it  by  the  addition  of  ten  volumes  of  99.8  per  cent -alcohol 
and  is  allowed  to  settle  in  tall  glass  cylinders.  The  supernatant 


50  CHEMICAL  STATICS 

fluid  is  then  syphoned  off  and  the  precipitate  is  washed  in  the 
same  volume  of  alcohol  as  that  employed  in  the  precipitation. 
This  washing  is  repeated,  again  employing  the  same  volume  of 
alcohol,  and  the  precipitate  is  allowed  to  steep  in  this  alcohol  for 
about  24  hours,  in  order  to  remove  all  adherent  or  combined 
acetic  acid.  The  alcohol  is  then  syphoned  off  and  the  precipitate 
is  washed  in  the  same  volume  of  ether  (distilled  over  sodium). 
This  washing  is  repeated.  The  ether  is  then  syphoned  off  and 
the  thick  suspension  of  protein  in  ether  thus  obtained  is  rapidly 
poured  into  a  hardened  filter,  the  filter  and  the  contained  sus- 
pension of  protein  in  ether  being  at  once  transferred  to  an  incu- 
bator and  the  filtration  continued  over  sulphuric  acid  at  40  degrees 
(to  avoid  condensation  of  atmospheric  moisture  on  the  filter). 

After  the  completion  of  filtration,  the  ether  which  has  filtered 
off  is  removed  from  the  incubator,  and  the  precipitate  is  allowed 
to  dry  for  24  hours.  The  protein  is  then  obtained  in  the  form 
of  chalky  cakes  which  are  very  readily  broken  up  into  impal- 
pable powder.  This  powder  is  passed  through  a  fine  sieve  and 
conserved  in  a  glass  stoppered  bottle. 

It  is  found  inadvisable  to  work  with  fewer  than  six  dozen  eggs 
at  one  time  as,  otherwise,  the  amount  of  precipitate  which  is 
finally  obtained  is  so  small  that  the  danger  of  excessive  caking 
and  partial  decomposition,  in  drying,  due  to  the  deposition  of 
traces  of  moisture  upon  the  filter,  is  very  great. 

Twenty-four  dozen  eggs  of  average  size  yield  about  40  grams  of 
ovomucoid. 

About  a  gram  of  ovomucoid,  prepared  in  this  manner,  was 
dissolved  in  about  100  cc.  of  N/2  hydrochloric  acid,  and  this 
solution  was  boiled  until  30  cc.  of  fluid  had  distilled  over.  This 
distillate  was  then  tested  for  acetic  acid.  It  contained  a  trace 
of  an  acid  of  the  fatty  series,  sufficient  to  yield  a  slight  coloration 
with  ferric  chloride,  but  insufficient  to  yield  a  precipitate  of  ferric 
hydrate  on  boiling,  or  to  yield  the  ethyl  acetate  test.* 

Ovomucoid  is  a  gluco-protein,  and  yields,  on  boiling  with 
strong  acid,  about  30  per  cent  of  glucosamin  (47)  (40)  (18)  (42) 
which,  according  to  Steudel  (42),  is  contained  in  the  molecule  not 
in  the  form  of  glucosamin  but  in  the  form  of  an  antecedent  which 
yields  glucosamin  on  treatment  with  strong  acids. 

*  The  solutions  of  ovomucoid  prepared  in  this  manner  have  themselves  a 
distinct  odor  of  ethyl  acetate. 


GELATIN  51 

11.  Gelatin  and  Deaminized  Gelatin.  —  The  only  practicable 
method  of  purifying  gelatin  which  has  been  devised  consists  in 
subjecting  the  best  qualities  of  commercial  gelatin  to  very  pro- 
longed dialysis  against  running  distilled  water  (4).     Deaminized 
gelatin  is  prepared  by  Blasel  and  Matula  (1)  by  the  following 
method : 

About  200  grams  of  the  best  commercial  gelatin  is  dissolved 
in  one  litre  of  warm  water.  To  this  solution  is  added  200  grams 
of  sodium  nitrite  dissolved  in  one  litre  of  water.  After  cooling, 
140  grams  of  glacial  acetic  acid  is  carefully  added.  The  mix- 
ture is  allowed  to  stand  for  twelve  hours  and  then  heated  on 
a  water-bath  for  two  hours.  The  deaminized  gelatin  is  then 
precipitated  by  the  addition  of  solid  ammonium  sulphate  and 
purified  by  prolonged  dialysis  (2  weeks)  against  running  distilled 
water. 

12.  Globin.  —  Globin  may  best  be  prepared  by  the  following 
modification  of  the  method  devised  by  Schulz  (39)  (37). 

A  thick  suspension  of  ox-corpuscles  is  obtained  by  centrif- 
ugalizing  freshly  defibrinated  ox-blood,  the  volume  of  the  sus- 
pension being  about  one-third  that  of  the  blood  from  which  it 
is  derived. 

After  pipetting  off  the  supernatant  serum  the  suspension  is 
diluted  to  the  original  volume  of  the  blood  from  which  it  was 
derived  by  the  addition  of  N/Q  NaCl  solution  and  the  centrif- 
ugalization  is  repeated,  the  supernatant  fluid  being  removed  as 
before.  This  is  repeated  six  times  in  order  to  free  the  corpuscles 
from  adherent  serum.  After  the  last  centrifugalization  the  cor- 
puscle suspension  is  not  again  diluted  with  NaCl. 

The  thick  suspension  of  corpuscles  which  is  thus  obtained  is 
diluted  to  ten  times  its  volume  by  the  addition  of  distilled  water. 
The  corpuscles  are  thus  "laked"  and  the  contained  haemoglobin 
is  discharged  into  the  water,  forming  a  clear  solution  which  is 
allowed  to  stand  in  tall  glass  vessels  for  twenty-four  hours  in 
order  to  permit  the  leucocytes  to  settle.  The  upper  portion  of 
the  fluid  is  then  decanted  and  employed,  the  lower  portion  of 
the  fluid  being  rejected. 

Two  and  one-half  litre  portions  of  this  fluid  are  placed  in  six- 
litre  bottles,  to  the  contents  of  each  bottle  are  added  56  cc.  of 
concentrated  HC1  (sp.  gr.  1.18)  and  the  mixture  is  thoroughly 
shaken  and  allowed  to  stand  at  room  temperature  for  one  hour. 


52  CHEMICAL  STATICS 

The  addition  of  the  acid  causes  a  flocculent  precipitate  to  appear 
and  the  mixture  turns  dark  brown.  Two  and  one-half  litres  of 
ether  are  then  added  to  each  bottle  and  the  contents  shaken 
thoroughly  until  they  attain  an  oily  consistency.  Rubber  stop- 
pers with  two  perforations  are  then  fitted  into  the  necks  of  the 
bottles.  Through  one  perforation  is  inserted  a  long  glass  tube 
reaching  to  the  bottom  of  the  bottle,  to  act  subsequently  as  an 
air  inlet,  and  through  the  other  is  inserted  a  short  tube  just  reach- 
ing to  the  bottom  of  the  stopper  and  provided  with  a  rubber 
tube  and  pinch-cock.  The  stoppers  are  then  tied  down  and  the 
bottles  quickly  inverted  and  allowed- to  stand  at  room  tempera- 
ture for  twenty-four  hours.  By  this  time  the  contents  of  the 
bottles,  if  the  temperature  of  the  room  is  not  too  warm,  should 
have  separated  into  two  layers,  an  upper,  jelly-like,  very  deeply 
colored  ether  layer  containing  the  greater  part  of  the  hsematin 
hydrochloride,  and  a  lower,  aqueous  layer  containing  the  globin 
to  some  extent  contaminated  by  hsematin  hydrochloride.  The 
latter  is  then  drawn  off  through  the  shorter  of  the  two  tubes 
inserted  through  the  stopper.* 

To  this  fluid  is  added  four  volumes  of  a  mixture  of  equal  parts 
by  volume  of  alcohol  and  ether.  A  light-colored  precipitate  is 
obtained,  leaving  the  fluid  very  deeply  colored.  This  precipi- 
tate is  collected  upon  a  hardened  filter,  washed  once  with  a  volume 
of  alcohol  equal  to  that  of  the  watery  fluid  from  which  it  was 
precipitated,  drained  and  then  scraped  off  the  paper  and  dissolved 
in  a  volume  of  JV/10  HC1  equal  to  that  of  the  fluid  from  which 
the  globin  was  precipitated.  This  solution  is  now  diluted  by 
the  addition  of  three  times  its  volume  of  distilled  water,  and 
20  per  cent  ammonia  solution  is  carefully  added  until  a  precipitate 
just  appears.  One  cubic  centimetre  of  strong  ammonia  per  litre 
is  then  added  and  the  dense,  flocculent  precipitate  is  collected 
on  a  filter.  The  precipitate  is  then  washed  in  large  volumes  of 
absolute  alcohol,  alcohol-ether  mixture  and  ether.  After  drain- 
ing in  a  dry  atmosphere  (preferably  within  an  incubator  over 
H2SO4)  the  precipitate  is  allowed  to  dry  for  24  hours  in  an  incu- 
bator over  H2S04,  then  pulverized  and  sifted  and  returned  to  the 
incubator  to  dry  over  H2SO4  for  one  week. 

*  The  separation  of  the  fluid  into  two  layers  is  sometimes  very  slow.  In 
such  cases  separation  may  be  hastened  by  centrifugalization. 


PROTAMINS  53 

13.  The  Protamins.  —  Kossel,  to  whose  investigations  we 
owe  our  comparatively  extensive  knowledge  of  these,  "the  sim- 
plest proteins,"  prepares  them  in  the  following  manner  (12). 

The  minced  ripe  testicles  of  herrings  (yielding  clupein),  salmon 
(yielding  salmin)  or  sturgeons  (yielding  sturin)  are  shaken  up  in 
water,  whereby  a  suspension  of  spermatozoa  is  obtained.  This 
fluid  is  coagulated  with  acetic  acid  and  the  precipitate  washed 
with  alcohol  and  ether  and  dried.  About  100  grams  of  the  dry 
mass  is  then  shaken  up  in  500  cc.  of  a  1  per  cent  solution  of 
H2S04  for  one-half  hour  and  filtered;  this  extraction  is  repeated 
six  times  and  the  extracts  are  mixed.  The  filtered  extract  is 
precipitated  with  three  times  its  mass  of  alcohol,  the  fluid  is 
syphoned  off  and  the  precipitate  is  dissolved  in  hot  water  and 
again  precipitated  with  alcohol. 

This  precipitate  of  protamin  sulphate  is  dissolved  in  about  one 
and  one-half  litres  of  hot  water  and  the  solution  is  allowed  to 
cool,  when  a  small  part  of  the  sulphate  separates  out  as  a  yellow 
or  brownish  colored  oil.  This  least  soluble  portion  of  the  pro- 
tamin sulphate  is  rejected.  The  supernatant  fluid  is  collected 
and  evaporated  to  a  small  volume  when  the  greater  part  of  the 
protamin  sulphate  separates  out  as  an  oil.  The  mixture  is  placed 
in  a  separatory  funnel  and  the  oil  drawn  off. 

This  preparation  may  be  freed  from  the  last  traces  of  associated 
nucleic  acid  in  the  following  manner:  It  is  dissolved  in  hot 
water  and  then  precipitated  with  sodium  picrate.  This  precipi- 
tate is  well  washed  with  water  and  then  freed  from  picric  acid 
by  shaking  it  up  with  ether  in  the  presence  of  an  excess  of  H2S04. 
The  protamin  sulphate  is  precipitated  out  of  the  sulphuric  acid 
solution  by  the  addition  of  alcohol.  This  precipitation  should  be 
repeated.  The  protamin  sulphate  should  now  come  down  as  a 
loose  white  precipitate.  If  the  precipitate  -has  a  glutinous  ap- 
pearance, the  solution  in  water  and  precipitation  by  alcohol 
should  be  repeated.  Finally,  the  precipitate  is  dried  at  110  to 
120°  C. 

According  to  Kossel,  clupein  and  salmin  are  identical,  the 
formula  of  the  sulphate  being  a  multiple  of  CsoHsTNiyOe,  2  H2SC>4. 
The  most  probable  formula  for  sturin  sulphate  is,  according  to 
the  same  author,  4  CseHegNigO:  +  11  H2S04. 

These  substances  are  strong  bases;  when  one  precipitates  the 
H2S04  out  of  a  solution  of  clupein  sulphate  by  the  addition  of 


54  CHEMICAL  STATICS 

the  equivalent  mass  of  baryta,  the  resultant  solution  of  free 
clupein  has  a  strongly  alkaline  reaction  (12)  (43). 

Salmin  carbonate  yields  an  alkaline  solution  owing  to  hydro- 
lytic  dissociation  (43).  Taylor  states  that  the  salmin  yielded  by 
the  Pacific  salmon  is  identical  with  that  which  is  yielded  by  the 
European  salmon. 

The  protamins  readily  form  " basic  salts"  in  which  the  pro- 
portion of  H2SO4  to  protein  is  less  than  in  the  fully  neutralized 
compound;  they  also  tend  to  combine  with  water  to  form  vari- 
ous hydrates.  Thus  salmin  sulphate  which  is  insufficiently  dried 
has  the  formula  C3oH57Ni706,  2  H2SO4  +  H2O. 


LITERATURE   CITED 

(1)  Blasel,  L.,  and  Matula,  J.,  Biochem.  Zeit.  58  (1914),  p.  417. 

(2)  Bosworth,  A.  W.,  Journ.  Biol.  Chem.  20  (1915),  p.  91. 

(3)  Bosworth,  A.  W.,  and  Van  Slyke,  L.  L.,  Journ.  Biol.  Chem.  14  (1913), 

p.  203;  19  (1914),  p.  67. 

(4)  Chiari,  R.,  Biochem.  Zeit.  33  (1911),  p.  167. 

(5)  Freund,  E.,  and  Joachim,  J.,  Zeit.  fiir  Physiol.  Chem.  36  (1902),  p.  407. 

(6)  Gruebler,  G.,  Journ.  Prakt.  Chem.  23  (1881),  p.  97. 

(7)  Hammarsten,  O.,  Ergeb.  der  physiol.  1  Abt.  1  (1902),  p.  330. 

(8)  Hammarsten,  O.,  "A  Text-book  of  Physiological  Chemistry,"  trans. 

by  J.  A.  Mandel,  6th  English  Edn.  New  York,  1911. 

(9)  Hardy,  W.  B.,  Journ.  of  Physiol.  33  (1905),  p.  330. 

(10)  Hopkins,  F.  Gowland,  and  Pinkus,  S.  N.,  Journ.  of  Physiol.  23  (1898), 

p.  130. 

(11)  Hoppe-Seyler,  F.,  Med.-chem.  Untersuch.  (1866),  p.  216. 

(12)  Kossel,  A.,  Zeit.  f.  physiol.  Chem.  25  (1898),  p.  165. 

(13)  Kosutany,  T.,  Journ.  Landw.  51  (1903),  p.  130. 

(14)  Kuhne,  W.,  "Lehrbuch  der  physiol.  Chem.,"  Leipzig  (1868),  pp.  168 

and  174. 

(15)  Lacqueur,  E.,  and  Sackur,  0.,  Beitr.  z.  chem.  Physiol.  und  Pathol.  3 

(1902),  p.  196. 

(16)  Marcus,  E.,  Zeit.  f.  physiol.  Chem.  28  (1899),  p.  559. 

(17)  Morner,  C.  T.,  Zeit.  f.  physiol.  Chem.  18  (1894),  p.  535. 

(18)  Muller,  F.,  Zeit.  f.  Biol.  42  (1901),  p.  468. 

;(19)   Osborne,  T.  B.,  Amer.  Chem.  Journ.  13  (1891),  p.  408;  14  (1892),  pp. 
212,  662. 

(20)  Osborne,  T.  B.,  Amer.  Chem.  Journ.  14  (1892),  p.  629. 

(21)  Osborne,  T.  B.,  "The  Proteins  of  the  Wheat  Kernel,"  Carnegie  Inst. 

Publ.,  Washington  (19CJY). 

(22)  Osborne,  T.  B.,  "The  Vegetable  Proteins,"  London  (1910). 

(23)  Osborne,  T.  B.,  and  .Campbell,  G.  F.,  Journ.  Amer.  Chem.  Soc.  22 

(1900),  p.  413. 


LITERATURE  CITED  55 

(24)  Osborne,  T.  B.,  and  Harris,  I.  F.,  Amer.  Journ.  of  Physiol.  17  (1906), 

p.  233. 

(25)  Plimmer,  H.  A.,  Journ.  Chem.  Soc.  London  (1908),  p.  1500. 

(26)  Preyer,  W.  T.,  "Die  Blutkrystalle,"  Jena,  1871. 

(27)  Quinan,  C.,  Univ.  of  California  Publ.  Pathol.  1  (1903),  p.  1. 

(28)  Reichert,  E.  T.,  and  Brown,  A.  P.,  Carnegie  Inst.  Publ.  Nr.  116,  Wash- 

ington (1909). 

(29)  Ritthausen,  H.,  Journ.  Prakt.  Chem.  23  (1881),  p.  481. 

(30)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  2  (1907),  p.  317. 

(31)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  2  (1907),  p.  337;  5  (1909), 

p.  493;  8  (1910),  p.  287. 

(32)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  13  (1909),  p.  469. 

(33)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  7  (1910),  p.  359. 

(34)  Robertson,  T.  Brailsford,  Journ.  Physical.  Chem.  14  (1910),  p.  377. 

(35)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  14  (1910),  p.  528. 

(36)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  14  (1910),  p.  709. 

(37)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  13  (1913),  p.  455. 

(38)  Schmidt,  A.,  Arch.  f.  (Anat.  und)  Physiol.  (1862),  p.  428. 

(39)  Schulz,  F.  N.,  Zeit.  f.  physiol.  Chem.  24  (1898),  p.  449. 

(40)  Seeman,  T.,  Arch.  f.  Verdanungskrankheiten.  Bd.  4  (1898),  cited  after 

Muller  (18). 

(41)  Soldner,  F.,  Landw.  Versuchsst.  35  (1881),  p.  351. 

(42)  Steudel,  H.,  Zeit.  f.  physiol.  Chem.  34  (1901),  p.  353. 

(43)  Taylor,  A.  E.,  Univ.  of  California  Publ.  Pathol.  1  (1904),  p.  7. 

(44)  Taylor,  A.  E.,  Journ.  Biol.  Chem.  1  (1906),  p.  345. 

(45)  Van  Slyke,  L.  L.,  and  Hart,  E.  B.,  Amer.  Chem.  Journ.  33  (1905),  p.  461. 

(46)  Van  Slyke,  L.  L.,  and  Van  Slyke,  D.  D.,  Amer.  Chem.  Journ.  38  (1907), 

p.  393. 

(47)  Zanetti,  C.  U.,  Ann.  di.  Chim.  E.  Farm.  (1897),  Nr.  12,  cited  after 

Steudel  (42). 


CHAPTER  III 
THE   QUANTITATIVE  ESTIMATION   OF  THE  PROTEINS 

1.  The  General  Principles  Underlying  the  Quantitative 
Estimation  of  Proteins.  —  The  extraordinary  bulk  of  the  protein 
molecule  in  comparison  with  the  molecules  of  the  simpler  sub- 
stances which  were  the  first  to  claim  investigation  by  analytical 
chemists,  places  unusual  difficulties  in  the  way  of  the  estimation 
of  proteins  by  the  traditional  methods  of  analytical  chemistry. 
Although  the  proteins  act  as  acids  and  bases,  yet  their  amphoteric 
character  seriously  interferes  with  direct  titration  by  acidimetric 
methods,  for  both  the  acidic  and  basic  functions  of  the  proteins 
are  weakened  and  modified  by  their  simultaneous  presence  in  the 
same  molecule,  and  it  is  but  rarely,  in  cases  such  as  those  afforded 
by  casein  and  the  various  members  of  the  protamin  group,  that 
the  predominance  of  the  one  function  is  sufficiently  great  to 
permit  of  its  utilization  in  the  determination  of  the  protein  by 
direct  titration  (1)  (18)  (32).  Even  in  these  cases  we  are  addi- 
tionally hampered  by  the  great  magnitude  of  the  combining- 
weight  of  the  protein,  leading  to  a  proportionate  enhancement 
of  the  normal  margin  of  acidimetric  inexactitude,  and  also  by  the 
fact  that  many  of  the  indicators  which  are  customarily  employed 
in  alkalimetry  or  acidimetry  form  compounds  with  proteins,  either 
ceasing  to  function  as  indicators,  or  displaying  novel  or  abnormal 
color-changes  or  even  directly  entering  into  and  disturbing  the 
very  equilibrium,  between  protein  on  the  one  hand  and  an  acid 
or  base  on  the  other  hand,  which  we  desire  to  measure. 

Since  the  nitrogen-content  of  the  proteins  is  high  and  nitrogen 
is  one  of  the  elements  which  we  are  able  to  determine  with  the 
greatest  precision,  nearly  all  the  efforts  of  analytical  chemists 
in  this  field  have  until  recently  been  aimed  at  reducing  the  esti- 
mation of  protein  to  an  estimation  of  nitrogen.  The  problem 
thus  resolved  itself  into  one  of  separating  the  protein  in  question 
from  other  proteins  and  from  nitrogenous  contaminations.  In 
cases  in  which  this  preliminary  isolation  is  not  difficult  of  attain- 

56 


QUANTITATIVE  ESTIMATION  57 

ment,  as  in  the  case  of  the  casein  in  milk,  no  more  accurate  and 
generally  reliable  method  of  procedure  has  yet  been  devised. 
Unfortunately  in  many,  if  not  in  the  majority  of,  instances  it  is 
the  fulfilment  of  this  preliminary  condition  of  complete  separa- 
tion from  nitrogenous  contaminations  which  presents  the  most 
serious  difficulties.  For  instance  the  most  valuable  reagent  for 
accomplishing  the  separation  of  various  members  of  the  globulin 
group  from  one  another  and  from  other  proteins  is  ammonium 
sulphate,  but  the  coagulum  which  is  produced  by  this  reagent, 
necessarily  employed  in  high  concentration,  is  heavily  contami- 
nated by  the  ammonium  salt  and  must  be  freed  therefrom  by 
very  prolonged  dialysis  before  it  can  be  utilized  for  a  determination 
of  nitrogen.  Prolonged  dialysis,  on  the  other  hand,  is  not  only 
time-consuming  but  also  involves  the  possibility  of  serious  errors 
arising  from  autohydrolysis  or  even  hydrolysis  due  to  enzymes 
or  to  bacterial  contamination,  due  to  the  protracted  period  during 
which  the  labile  protein  must  be  exposed  to  the  action  of  water. 
Any  alternative  method  of  freeing  the  protein  from  contamina- 
tion by  ammonium  salts,  such  as  resolution  followed  by  recoagu- 
lation  with  some  other  reagent,  is  attended  with  many  difficulties 
and  uncertainties  attributable  in  large  proportion  to  the  modi- 
fications of  the  actions  of  coagulating  agents  which  are  brought 
about  by  the  saline  contamination  which  we  desire  to  remove. 

The  high  combining-weight  of  the  proteins  attributable  to  the 
mass  of  their  molecules,  is,  as  we  have  seen,  the  chief  obstacle 
to  the  application  of  chemical  methods  in  their  quantitative 
estimation.  In  the  application  of  physical  methods  for  this  pur- 
pose, however,  the  bulk  of  the  protein  molecule,  far  from  being 
a  disadvantage,  is  frequently  a  positive  advantage  and  may  indeed 
enable  us  to  employ  physical  methods  of  estimation  not  ordi- 
narily applicable  to  the  smaller  molecules  of  the  majority  of 
inorganic  or  the  simpler  classes  of  organic  substances,  for  the 
great  majority  of  the  physical  properties  of  a  substance  are 
determined  by  the  mass  or  volume  of  its  molecules  and  those 
properties  which  are  magnified  by  increasing  mass  of  the  molecule 
are  of  course  displayed  exceptionally  prominently  by  the  proteins. 

It  appears  extremely  probable  therefore  that  in  the  future 
we  will  come  to  rely  more  and  more  upon  physical  methods  for 
the  estimation  of  the  characteristically  bulky  molecules  of  col- 
loidal substances.  Many  attempts  have  been  made  to  estimate 


58  CHEMICAL  STATICS 

the  protein-content  of  fluids  by  their  optical  rotation.  This 
particular  physical  property  of  the  proteins  was,  however,  a 
most  unfortunate  choice,  since  it  is  one  of  the  few  physical  prop- 
erties which  is  independent  of  the  absolute  mass  or  volume  of 
the  molecules  displaying  it,  and  the  specific  rotatory  power  of 
proteins  is  extremely  low  in  comparison  with  that  of  a  large 
proportion  of  optically  active  substances  of  much  smaller  molec- 
ular dimensions. 

In  recent  years  two  methods  of  estimating  protein,  based  upon 
the  measurement  of  physical  qualities  of  their  solutions  or  sus- 
pensions, have  been  suggested  and  applied  to  specific  problems 
in  very  considerable  detail.  These  are  the  nephelometric  method, 
based  upon  measurements  of  opacity,  and  the  refractometric 
method.  Both  methods  are  capable  of  attaining  a  high  degree 
of  accuracy  and  may  be  rendered  to  a  great  extent  independent 
of  contamination  by  non-protein  substances.  It  therefore  ap- 
pears probable  that  they  may  eventually  prove  applicable 
to  the  estimation  of  a  variety  of  proteins  under  conditions  which 
would  preclude,  or  render  exceedingly  difficult,  reduction  of  the 
estimation  to  a  determination  of  nitrogen. 

2.  The  Nephelometric  Method  of  Estimating  Proteins.  — 
This  method,  which  we  owe  to  Kober  and  his  collaborators  (6) 
(7)  (8)  (9)  (10),  depends  upon  the  measurement  of  the  relative 
opacities  of  suspensions  of  coagulated  proteins,  the  measurement 
of  relative  opacity  being  carried  out  in  a  modified  form  of  color- 
imeter. A  full  description  of  this  instrument  will  be  found  in  a 
recent  article  by  Kober  and  Graves  (9). 

The  success  of  the  nephelometric  method  depends  upon  the 
discovery  of  specific  coagulating  agents  which  will  bring  the 
protein  into  stable  suspension  in  masses  of  approximately  uni- 
form, or  at  least  reproducible,  magnitude,  of  sufficiently  large 
diameter  to  scatter  incident  light-rays  and  insufficiently  large  to 
result  in  instability  of  the  suspension.  The  discovery  of  the  most 
suitable  coagulating  agent  for  each  protein  and  under  each  set 
of  modifying  circumstances  constitutes,  indeed,  the  chief  diffi- 
culty of  the  method,  but,  once  the  correct  coagulant  is  found 
and  the  circumstances  attending  its  employment  have  been 
standardized,  the  method  is  susceptible  of  a  very  high  degree 
of  accuracy,  far  surpassing  indeed  that  attainable  through  the 
estimation  of  nitrogen. 


NEPHELOMETRIC  METHOD  59 

The  coagulating  reagent  employed  by  Kober  is  sulpho- 
salicylic  acid.  The  following  are  the  details  of  the  method 
as  applied  by  Kober  to  the  estimation  of  the  various  proteins  in 
milk  (8). 

Five  cubic  centimetres  of  milk  are  carefully  measured  into  a 
250-cc.  graduated  flask  and  after  adding  200  cc.  of  distilled  water 
and  10  cc.  of  decinormal  sodium  hydroxide  solution,  water  is 
added  to  the  mark  and  the  solution  is  thoroughly  shaken.  Ten 
cubic  centimetres  of  this  mixture  is  then  placed,  together  with 
2  cc.  of  ether  (which  has  previously  been  washed  with  a  10  per 
cent  aqueous  solution  of  sodium  hydroxide)  in  a  centrifuge  tube 
which  is  then  tightly  stoppered  with  a  cork  and  vigorously  shaken. 
After  allowing  the  mixture  to  stand  until  the  layers  have  sepa- 
rated, or  after  centrifuging  for  one  to  two  minutes,  the  cork  is 
removed  and  5  cc.  of  the  aqueous  layer  is  withdrawn.  This  is 
done  by  closing  the  top  of  the  pipette  with  the  finger  and  insert- 
ing it  quickly  into  the  centrifuge  tube.  If  this  is  done  correctly 
the  ether  solution  will  not  contaminate  the  sample.  This  5  cc. 
of  the  aqueous  layer  is  then  diluted  in  a  volumetric  flask  to  ex- 
actly 50  cc. 

The  milk  treated  in  this  way  has  an  almost  inappreciable  tur- 
bidity, not  more  than  1.6  per  cent  of  the  turbidity  which  is  sub- 
sequently produced  by  the  coagulating  agent.  Such  residual 
turbidity  as  it  possesses  is  almost  exactly  equal  to  that  of  the 
standard  solution  of  casein  with  which  it  is  subsequently  com- 
pared, and  is  therefore  without  influence  upon  the  accuracy  of 
the  comparison  with  the  standard. 

To  10  cc.  of  this  solution  is  now  added  10  cc.  of  a  3  per  cent 
solution  of  sulphosalicylic  acid  and  the  suspension  of  coagulated 
milk  protein  which  is  thus  obtained  is  matched  in  the  nephel- 
ometer  with  the  following  standard:  One  volume  (5  cc.)  of  a 
0.01  per  cent  casein  solution  to  which  is  added  two  volumes 
(10  cc.)  of  3  per  cent  sulphosalicylic  acid. 

The  volume  of  solution  employed  is  not  a  correct  aliquot  of 
the  original  sample  of  milk,  owing  to  the  fact  that  in  the  extrac- 
tion with  ether  some  ether  is  dissolved  by  the  aqueous  layer  and 
some  water  by  the  ether  layer.  A  determination  of  the  volume 
increase  of  a  solution  of  sodium  caseinate  shaken  up  with  ether 
shows  that  10  cc.  of  the  clear  aqueous  layer  of  defatted  diluted 
milk  is  equal  to  9.1  cc.  of  the  diluted  milk  before  extraction  with 


60  CHEMICAL  STATICS 

ether.  Correction  must  be  made  for  this  change  of  volume  in 
the  estimate  of  protein-content. 

In  order  to  obtain  the  casein-content  of  the  milk,  the  casein 
from  a  fresh  portion  of  milk  is  precipitated  in  accordance  with  the 
"official"  method  by  the  addition  of  acetic  acid,  and  the  opacity 
of  the  suspension  obtained  in  an  aliquot  part  of  the  filtrate,  by 
adding  four  volumes  of  3  per  cent  sulphosalicylic  acid  solution, 
is  determined  nephelometrically.  This  determination  yields  an 
estimate  of  the  " globulin  and  albumin"  fraction  which,  sub- 
tracted from  the  " total  proteins"  estimated  above,  yields  the 
amount  of  casein  precipitable  by  the  "  official "  method. 

A  modification  of  this  method  of  protein  estimation  has  been 
utilized  by  Pfeiffer,  Kober  and  Field  (10)  in  the  estimation  of 
the  globulins  and  total  proteins  in  cerebrospinal  fluid. 

It  will  be  seen  that  the  chief  difficulty  attaching  to  the  nephelo- 
metric  method  arises  from  the  necessity  of  achieving  constant 
conditions  of  coagulation,  a  difficulty  which  is  especially  serious 
when  the  coagulating  process  has  to  be  superadded  to  the  pro- 
cedures incident  to  the  separation  from  one  another  of  the  vari- 
ous proteins  in  a  mixture  in  which  it  is  desired  to  determine 
each  protein  separately.  This  difficulty  does  not  attach  to  the 
refractometric  method  described  below,  in  which,  once  the  sepa- 
ration of  the  various  protein  fractions  has  been  achieved,  no 
further  procedures  other  than  dilution,  etc.,  are  necessitated 
before  a  determination  can  be  made.  On  the  other  hand,  the 
refractometric  method  does  not  permit  the  extreme  accuracy 
which  may  be  attained  with  the  nephelometric  method  under 
favorable  conditions. 

3.  The  Refractometric  Method  of  Estimating  Proteins.  — 
This  method,  originally  employed  by  Reiss  (11)  (12)  (13)  (14) 
(15)  (16)  for  the  estimation  of  the  total  proteins  in  blood-serum, 
has  been  modified  by  the  author  so  as  to  render  it  applicable  to 
the  separate  determination  of  the  non-proteins,  globulins,  albu- 
mins and  total  proteins  in  a  little  over  1.5  cc.  of  blood-serum  (24). 
The  following  are  the  details  of  the  method : 

(a)  Estimation  of  the  non-proteins. 

Glass  tubes  25  cm.  long,  having  an  inside  diameter  of  5  mm.  and 
walls  about  1  mm.  thick,  are  sealed  at  one  end.  It  is  well  to  blow 
gently  into  the  tube  while  the  sealed  end  is  still  soft,  thus  making 
the  contour  of  the  bottom  of  the  tube  hemispherical  and  dimin- 


REFRACTOMETRIC   METHOD  61 

ishing  the  tendency  to  crack  on  cooling.  Into  one  of  these  tubes, 
which,  has  been  carefully  cleaned  and  dried,  is  now  introduced 
exactly  1  cc.  of  serum  with  the  aid  of  an  accurately  calibrated 
pipette  with  a  capillary  tip.  Such  pipettes  may  be  prepared  by 
taking  lengths  of  narrow-bore  glass  tubing  drawn  out  to  a  capil- 
lary at  one  end,  introducing  1  cc.  of  mercury,  and  marking  with 
a  diamond  the  extremities  of  the  mercury  column.  The  mercury 
is  then  delivered  into  another  similar  tube,  which  is  similarly 
marked,  and  the  operation  repeated  until  the  desired  number  of 
pipettes  is  obtained.  Prepared  in  this  way  the  pipettes  all  deliver 
(between  the  marks)  the  same  volume  of  fluid,  and  if  this  is  the 
case  the  exact  volume  employed  is  immaterial  provided  it  is  in 
the  neighborhood  of  1  cc. 

In  delivering  the  serum,  wetting  the  upper  part  of  the  tube  and 
the  formation  of  air-bubbles  should  be  carefully  avoided. 

The  serum  having  been  introduced,  with  another  pipette  cali- 
brated against  the  first,  deliver  1  cc.  of  N/25  acetic  acid  solution, 
which  may  be  made  up  with  sufficient  accuracy  by  diluting  4  cc. 
of  glacial  acetic  acid  to  1750  cc. 

A  short  length  of  thick  platinum,  silver,  or  nickel  wire  is  now 
introduced  into  the  tube  and  the  upper  end  is  sealed  off  in  a  flame, 
taking  care  not  to  heat  the  contents.  After  cooling,  the  tube  is 
shaken  and  the  short  length  of  wire  contained  in  it  brings  about 
a  thorough  admixture  of  the  contents.  The  tube  is  now  placed 
in  a  beaker  of  cold  water  of  such  a  depth  as  to  immerse  the  top 
of  the  contained  column  of  liquid  several  millimeters  below  the 
surface.  It  is  well  to  rest  the  bottom  of  the  tube  upon  a  wad  of 
glass  wool  or  a  piece  of  wire  gauze  to  avoid  the  cracking  of  the 
tube  by  bumping  during  the  subsequent  boiling.  The  water  is 
now  slowly  heated  to  boiling  and  allowed  to  boil  energetically 
for  exactly  two  minutes.  The  tube  is  then  removed  from  the 
boiling  water  and  allowed  to  cool  to  room  temperature. 

When  cool  the  tube  is  broken  open  a  little  above  the  surface 
of  its  contents  and  the  coagulum  is  broken  up.  This  is  best 
accomplished  with  the  aid  of  a  platinum  wire  about  0.6-0.7  mm. 
in  diameter  and  provided  with  several  slight  bends.  This  is 
inserted  into  the  tube  and  the  upper  end  twirled  between  the 
thumb  and  forefinger.  The  fluid  and  the  coagulum  are  now 
separated  by  centrifugalization,  the  fluid  is  withdrawn  by  the 
aid  of  a  dry,  clean  pipette,  and  the  refractive  indices  of  the  fluid 


62  CHEMICAL  STATICS 

and  of  N/5Q  acetic  acid  solution  (prepared  by  diluting  the  N/25 
acetic  acid  used  above  to  exactly  one-half  with  distilled  water) 
are  determined  simultaneously.  By  determining  the  refractive 
indices  of  the  fluid  and  of  JY/50  acetic  acid  simultaneously,  the 
necessity  for  regulation  of  the  temperature  at  which  the  readings 
are  made  is  obviated,  for  although  the  absolute  values  of  the 
refractive  indices  are  affected  by  temperature,  the  differences 
between  them  are  independent  of  the  temperature  at  which  they 
are  determined  (19).  In  carrying  out  a  large  number  of  esti- 
mations, however,  it  is  necessary  to  eliminate  the  possible  error 
due  to  progressive  changes  in  the  temperature  of  the  dark-room  by 
redetermining  the  refractive  index  of  the  solvent  (in  this  instance 
JV/50  acetic  acid)  at  frequent  intervals. 

The  refractive  index  of  a  1  per  cent  solution  of  NaCl  is  0.00160 
greater  than  that  of  distilled  water.  The  refractivities  of  1  per 
cent  solutions  of  other  inorganic  salts  and  of  glucose  and  urea  are 
of  very  similar  magnitude.  To  within  a  sufficient  degree  of 
accuracy,  therefore,  the  percentage  of  non-protein  substances 
in  the  serum  may  be  estimated  by  dividing  their  refractivity 
by  the  factor  0.00160.  The  result  must  be  multiplied  by  2, 
because  the  serum  has  been  diluted  to  one-half  with  N/25  acetic 
acid. 

For  the  determination  of  the  refractive  index,  I  employ  a 
Pulfrich  refractometer,  which  reads  the  angle  of  total  reflection 
to  within  one  minute.  A  sodium  flame  is  used  as  the  source  of 
light.  The  refractive  index  may  be  estimated  from  the  angle 
of  total  reflection  with  the  aid  of  the  table  which  is  supplied  with 
the  instrument. 

When  it  is  desired  to  make  a  number  of  successive  determina- 
tions, the  cup  of  the  refractometer  should  be  carefully  dried  with 
absorbent  cotton  and  lens  paper  before  a  new  sample  of  fluid  is 
introduced. 

(b)  Estimation  of  the  albumins. 

Glass  tubes  9-10  cm.  long  are  prepared,  having  an  inside  diam- 
eter of  5  mm.  and  one  end  closed.  Tubes  which  have  been  em- 
ployed in  the  estimation  of  the  non-protein  constituents,  after 
having  been  cleaned  and.  dried,  may  be  cut  down'to  the  proper 
length  and  utilized  for  this  purpose.  Small  corks  or  short  pieces 
of  glass  tubing  sealed  at  one  end,  the  sealed  end  being  pressed 
against  the  open  end  of  the  longer  tubes  and  held  in  position  by 


REFRACTOMETRIC  METHOD  63 

short  pieces  of  rubber  tubing,  are  employed  as  stoppers.  Into 
one  of  these  tubes  is  introduced,  with  the  aid  of  a  pipette  similar 
to  that  described  above,  0.5  cc.  of  a  saturated  solution  of  ammo- 
nium sulphate  (prepared  by  dissolving  an  excess  of  ammonium 
sulphate  in  hot  water,  allowing  the  excess  to  crystallize  out  on 
cooling  and  then  removing  the  fluid  from  the  subnatant  crystals 
and  keeping  in  a  well-stoppered  container).  With  another  pi- 
pette which  has  been  calibrated  against  the  first,  introduce  0.5  cc. 
of  serum,  drop  in  a  piece  of  platinum,  silver  or  nickel  wire,  affix 
the  stopper  and  shake  thoroughly  with  as  little  delay  as  possible. 
It  is  necessary  to  introduce  the  ammonium  sulphate  first,  as  other- 
wise, being  of  greater  specific  gravity  than  the  serum,  it  sinks 
through  the  serum,  portions  of  which  are  thus  exposed  for  some 
time  to  ammonium  sulphate  of  higher  concentration  than  one- 
half  saturated.  This  leads  to  a  precipitation  of  albumins  which 
do  not  readily  redissolve,  and  the  results  obtained  are  erroneous 
and  irregular.  If  the  ammonium  sulphate  is  introduced  first 
the  serum  floats  on  the  top  of  it  and  energetic  shaking  brings 
about  almost  immediate  admixture  of  the  serum  and  the  reagent. 
It  is  well,  while  shaking,  to  hold  the  thumb  against  the  bottom 
of  the  tube,  thus  diminishing  the  danger  of  cracking  the  tube 
by  the  impacts  of  the  heavy  piece  of  wire. 

The  tube,  with  the  stopper  still  affixed,  is  now  centrifuged. 
The  precipitate  soon  settles  and  sufficient  supernatant  fluid  may 
,be  drawn  off  to  fill  the  tip  of  a  pipette  and  the  space  between 
two  marks  known  to  hold  about  0.25  cc.  (prepared  as  above). 
This  quantity  of  the  supernatant  fluid  is  delivered  into  another 
clean,  dry  tube  of  the  type  employed  in  the  precipitation,  and 
0.25  cc.  of  distilled  water  is  added  with  the  aid  of  a  pipette  cali- 
brated against  the  first.  A  piece  of  wire  is  dropped  in,  a  clean 
stopper  affixed,  and  the  contents  are  shaken.  We  now  deter- 
mine the  refractive  index  of  this  fluid  and  that  of  one-quarter 
saturated  ammonium  sulphate  solution  prepared  (and  kept  as  a 
stock  solution)  by  mixing  equal  volumes  of  saturated  ammonium 
sulphate  solution  and  distilled  water  and  adding  to  this  mixture 
an  equal  volume  of  distilled  water.  The  difference  between  these 
refractive  indices  represents  one-fourth  of  the  combined  refrac- 
tivities  of  the  albumins  and  of  the  non-protein  constituents. 
Multiplying  by  4,  therefore,  and  subtracting  the  refractivity 
of  the  non-protein  constituents,  we  have  the  refractivity  of  the 


64  CHEMICAL  STATICS 

albumins.  Dividing  this  by  0.00177,  we  obtain  the  percentage 
of  albumin  in  the  serum.  It  is  necessary  to  dilute  the  ammonium 
sulphate  solution  of  albumins  because  the  refractivity  of  proteins 
in  half -saturated  ammonium  sulphate  solutions  is  abnormal  (21). 

(c)  Estimation  of  the  globulins. 

Determine  the  refractive  index  of  the  serum  and  that  of  dis- 
tilled water.  Subtracting  from  the  difference  the  known  refrac- 
tivity of  the  non-proteins  and  the  known  refractivity  of  the 
albumins,  we  obtain  the  refractivity  of  the  globulins.  Dividing 
this  by  0.00229  we  obtain  the  percentage  of  globulin  in  the  serum. 
Adding  together  the  percentages  of  albumins  and  globulins  we 
obtain  the  percentage  of  total  proteins. 

The  following  is  an  illustrative  determination,  employing  ox- 
serum  obtained  by  whipping  and  centrifuging  freshly-drawn 
blood : 

Refractive  index  of  the  fluid  obtained  after  acidifying  with 

acetic  acid  and  boiling  (3  determinations) 1 . 33024 

Refractive  index  of  AT/50  acetic  acid 1 . 32902 

Difference  X  2 0.00244 

Estimated  concentration  of  non-proteins  =  fff  =  1.5  per  cent. 

Refractive  index  of  the  fluid  obtained  after  precipitation  of  the  globulins 
and  subsequent  dilution: 

1st  determination 1 . 35189 

2nd  determination 1 . 35172 

3rd  determination 1 . 35181 

4th  determination 1 . 35189 

Refractive  index  of  |  saturated  (NH4)2SO4 1 . 34932 

Differences  X  4: 

1st  determination 0.01028 

2nd  determination 0.00960 

3rd  determination 0.00996 

4th  determination 0.01028 

Refractivity  of  the  albumins: 

1st  determination  0.01028  -  0.00244  =  0.00784 
2nd  determination  0.00960  -  0.00244  =  0.00716 
3rd  determination  0.00096  -  0.00244  =  0.00752 
4th  determination  0.01028  -  0.00244  =  0.00784 
Average  0.00759 

Concentration  of  albumins: 

1st  determination  =  ^  =  4.4  per  cent 
2nd  determination  =  £rf  =  4.1  per  cent 
3rd  determination  =  £ff  =4.3  per  cent 
4th  determination  =  ty$  =  4.4  per  cent 
Average  4 . 3  per  cent 


REFRACTOMETRIC  METHOD  65 

Refractive  index  of  the  serum 1 . 34686 

Refractive  index  of  distilled  water 1 . 32887 

Difference 0.01799 

Refractivity  of  the  proteins  =  0.01799  -  0.00244  =  0.01555 
Refractivity  of  the  globulins  =  0.01555  -  0.00759  =  0.00796 
Concentration  of  globulins  =  Iff  =  3.5  per  cent 
Concentration  of  total  proteins  =  3.5  +  4.3  =  7.8  per  cent 

Since  the  percentage  of  total  proteins  is  dependent  upon  the 
dilution  of  the  blood  and  is  therefore  highly  variable,  while  the 
relative  proportions  of  globulin  and  albumin  are  much  more 
constant,  it  is  convenient  to  express  these  in  percentages  of  the 
total  proteins.  Thus  in  the  above  result,  taking  the  total  pro- 
teins as  100,  we  find  that  the  globulins  formed  45  per  cent  and 
the  albumins  55  per  cent  of  the  total  proteins. 

This  method  has  been  applied  to  the  study  of  the  proteins  in 
human  blood-serum  under  normal  conditions  and  a  variety  of 
disease-conditions  by  A.  H.  Rowe  (25)  (26)  (27)  (31).  It  has 
also  been  applied  to  the  study  of  the  changes  in  the  ratio  of 
globulin  to  albumin  in  blood-serum  during  immunization  and 
infection  by  Righetti  (17),  Hurwitz  and  Meyer  (4)  and  Schmidt 
(28).  The  globulins,  albumins  and  non-proteins  in  the  blood- 
sera  of  a  variety  of  mammals  and  birds  have  also  been  estimated 
refractometrically  and  characteristic  differences  found  in  the 
relative  proportions  of  these  constituents  in  widely-separated 
classes  of  animals  (2)  (3)  (5)  (23)  (30)  (33)  (34). 

The  refractometric  method  has  also  been  applied  to  the  esti- 
mation of  casein  in  milk  (20)  and  to  the  estimation  of  the  diges- 
tive activity  of  proteolytic  ferments  (22)  (29). 


LITERATURE   CITED 

(1)  Arny,  H.  V.,  and  Pratt,  T.  M.,  Amer.  Journ.  of  Pharmacy  78  (1906), 

p.  121. 

(2)  Briggs,  R.  S.,  Journ.  Biol.  Chem.  20  (1914),  p.  7. 

(3)  Buck,  L.  W.,  Journ.  of  Pharmacology  and  Exper.  Therap.  5  (1913), 

p.  553. 

(4)  Hurwitz,  S.  H.,  and  Meyer,  K.  F.,  Journ.  Exper.  Med.  24  (1916),  p. 

515. 

(5)  Jewett,  R.  M.,  Journ.  Biol.  Chem.  25  (1916),  p.  21. 

(6)  Kober,  P.  A.,  Journ.  Biol.  Chem.  13  (1912),  p.  485. 

(7)  Kober,  P.  A.,  Journ.  Amer.  Chem.  Soc.  35  (1913),  p.  290. 

(8)  Kober,  P.  A.,  Journ.  Amer.  Chem.  Soc.  35  (1913),  p.  1585. 


66  CHEMICAL  STATICS 

(9)   Kober,  P.  A.,  and  Graves,  S.  S.,  Journ.  Indust.  and  Engineering  Chem. 
7  (1915),  p.  843. 

(10)  Pfeiffer,  J.  A.  F.,  Kober,  P.  A.,  and  Field,  C.  W.,  Proc.  Soc.  Exper.  Biol. 

and  Med.  12  (1915),  p.  153. 

(11)  Reiss,  E.,  Beitr.  z.  Chem.  Physiol.  und  Pathol.  4  (1904),  p.  150. 

(12)  Reiss,  E.,  Arch.  f.  exper.  Path,  und  Pharm.  51  (1904),  p.  18. 

(13)  Reiss,  E.,  Miinchener  Med.  Wochenschr.  55  (1908),  p.  1853. 

(14)  Reiss,  E.,  Jahrb.  f.  Kinderheilkunde  70  (1909),  p.  174. 

(15)  Reiss,  E.,  Ergeb.  d.  inn.  Med.  und  Kinderh.10  (1913),  p.  531. 

(16)  Reiss,  E.,  Deutsch.  Arch.  f.  Klin.  Med.  117  (1915),  p.  175. 

(17)  Righetti,  H.,  University  of  California  Publ.  Pathol.  2  (1916),  p.  205. 

(18)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  2  (1907),  p.  371. 

(19)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  13  (1909),  p.  469. 

(20)  Robertson,  T.  Brailsford,  Journ.  Indust.  and  Engineering  Chem.  1 

(1909),  p.  723. 

(21)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  11  (1912),  p.  179. 

(22)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  12  (1912),  p.  23. 

(23)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  13  (1912),  p.  325. 

(24)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  22  (1915),  p.  233. 

(25)  Rowe,  A.  H.,  Journ.  of  Laboratory  and  Clin.  Med.  1  (1916),  p.  439. 

(26)  Rowe,  A.  H.,  Arch.  Int.  Med.  18  (1916),  p.  455. 

(27)  Rowe,  A.  H.,  Arch.  Int.  Med.  19  (1917),  p.  354. 

(28)  Schmidt,  E.  S.,  and  C.  L.  A.,  Journ.  of  Immunology  2  (1917),  p.  343. 

(29)  Schorer,  G.,  "Ueber  refraktometrische  Pepsinbestimmungen "  aus  der 

med.  Klinik  der  Universitat  Bern  (1908). 

(30)  Thompson,  W.  B.,  Journ.  Biol.  Chem.  20  (1914),  p.  1. 

(31)  Tranter,  C.  L.,  and  Rowe,  A.  H.,  Journ.  Amer.  Med.  Assn.  65  (1915), 

p.  1433. 

(32)  Van  Slyke,  L.  L.,  and  Bosworth,  A.  W.,  Journ.  Indust.  and  Engineering 

Chem.  1  (1909),  p.  768. 

(33)  Wells,  C.  E,,  Journ.  Biol.  Chem.,  15  (1913),  p.  37. 

(34)  Woolsey,  J.  H.,  Journ.  Biol.  Chem.  14  (1913),  p.  433. 


CHAPTER  IV 
THE   COMPOUNDS   OF  THE  PROTEINS 

1.  The  Amphoteric  Character  of  the  Proteins.  —  The  fact  that 
one  and  the  same  protein  can  combine  either  with  a  base  or  with 
an  acid  appears  to  have  been  first  clearly  stated  by  Platner  (33) 
in  1866.  The  term  "amphoteric"  which  is  now  used  to  desig- 
nate substances  possessing  this  power  is  due  to  Bredig  (4). 

The  source  of  this  dual  combining-capacity  of  the  proteins 
has  been  discussed  in  Chap.  I.  In  1886,  Strecker  (51)  pointed 
out  that  amino-acids  can,  like  other  substituted  ammonias,  bind 
acids  by  their  —  NH2  groups,  or,  as  we  now  know,  through  the  trans- 
formation of  trivalent  into  pentavalent  nitrogen;  while  they  can 
also  bind  bases  in  virtue  of  their  possession  of  a  —  COOH  group. 
It  is  probable  that  in  most  cases  a  proportion  of  the  amino- 
acid,  when  dissolved  in  water,  combines  with  water  to  form  a 
compound  analogous  to  ammonium  hydrate,  which  is  in  chemi- 
cal equilibrium  with  the  unhydrated  form  also  present  in  the 
solution.  Thus  amino-acetic  acid, 

NH2 


COOH 

when  dissolved  in  water,  is  believed  to  partially  undergo  the 
reaction 

/NH2  /NH3.OH 

CH>'  +  H.OH  =  CH</ 

XCOOH  XCOOH 

the  trivalent  nitrogen  becoming  pentavalent,  and  the  molecule 
splitting  off  both  hydrogen  and  hydroxyl  ions  in  definite  pro- 
portions. As  I  have  stated,  however,  the  transformation  is  not 
complete,  nor  is  it  by  any  means  an  easy  matter,  as  a  rule,  to 
determine  its  extent  (25)  (39). 

Nevertheless,  as  I  have  pointed  out  in  Chap.  I  and  as  will  be 
more  clearly  revealed  in  the  chapters  dealing  with  the  electro- 
chemistry of  the  proteins,  it  is  very  improbable  that  terminal 
—  NH3OH  or  COOH  groups  in  the  proteins  molecule  are  involved 

67 


68  CHEMICAL  STATICS 

in  the  union  of  the  majority  of  proteins  with  inorganic  acids  and 
bases.  The  active  agents  in  accomplishing  these  unions  are, 
more  probably,  —  COH.N  —  groups  within  the  protein  molecule. 

Whatever  may  be  the  method  of  union  between  inorganic  sub- 
stances and  the  proteins,  however,  there  is  now  no  longer  any 
doubt  that  it  is  of  very  general  occurrence.  The  methods  which 
have  been  employed  by  various  investigators  to  demonstrate 
the  existence  of  compounds  of  the  proteins  with  inorganic  sub- 
stances are  very  diverse  in  nature,  but  they  may  be  conveniently 
classed  as  direct  and  indirect.  The  direct  methods  consist  in  the 
precipitation  of  the  (supposedly)  unaltered  compound  by  appro- 
priate reagents  (in  the  case  of  compounds  such  as  those  with  the 
heavy  metals,  which  are  insoluble  in  water,  addition  of  a  reagent 
is  of  course  unnecessary),  followed  by  the  elementary  analysis 
of  the  precipitate,  or  in  the  analysis  of  soluble  compounds  of 
proteins  with  substances  which,  when  uncombined,  are  insoluble 
in  water.  The  indirect  methods  may  be  variously  designated: 
the  indirect  method  of  precipitation,  the  method  of  electrical 
conductivity,  the  cryoscopic  method,  the  potentiometric  method, 
the  method  of  catalysis,  the  indicator  method  and  the  method 
of  " masking"  the  physiological  effects  of  inorganic  substances 
by  the  addition  of  proteins  to  their  solutions.  The  results  ob- 
tained by  these  methods  will  be  considered  separately. 

2.  The  Direct  Method  of  Demonstrating  the  Existence  of 
Protein  Compounds  by  Precipitation  or  Coagulation.  —  This, 
which  is  the  method  most  frequently  employed,  consists,  usually, 
in  precipitating  or  coagulating  the  protein  salt  by  the  addition 
of  suitable  reagents,  the  reagent  which  is  most  commonly  em- 
ployed being  alcohol. 

The  very  important  question  immediately  suggests  itself, 
whether  or  not  the  reagent  which  is  employed  for  this  purpose 
precipitates  the  protein  compound  unaltered,  as  it  exists  in  the 
solution,  or  whether  the  compound  is  altered  in  the  process  of 
precipitation.  On  the  whole  it  may  be  said  that  too  little  atten- 
tion has  been  given  to  this  question  by  investigators  who  have 
employed  this  method  and  that  in  consequence  we  possess  very 
little  accurate  information  on  the  subject. 

It  appears,  however,  that  alcohol  may  be  relied  upon,  at  all 
events  within  certain  limits,  to  precipitate  the  salts  of  some 
proteins  with  bases  in  an  unaltered  condition,  that  is,  containing 


DIRECT  METHOD  OF  PRECIPITATION  69 

the  same  relative  proportion  of  base  to  protein  as  the  pre-existing 
compound  in  aqueous  solution.  Thus  Van  Slyke  and  Hart  (52) 
employed  alcohol  to  precipitate  calcium  caseinate,  and  they 
found  that  the  precipitate  from  solutions  neutral  to  litmus  con- 
tained exactly  the  quantity  of  calcium  (uncombined  with  hydro- 
chloric acid)  which  corresponds  with  the  quantity  of  calcium 
hydrate  which  casein  will  neutralize  to  litmus  while  in  aqueous 
solution.  Similarly  the  precipitate  from  solutions  neutral  to 
phenolphthalein  contained  the  calculated  quantity  of  calcium. 
As  we  shall  see  in  considering  the  electrochemical  phenomena 
which  accompany  the  coagulation  of  the  caseinates  by  alcohol, 
coagulation  of  these  salts  through  the  addition  of  alcohol  to  their 
aqueous  solutions  is  preceded  by  a  profound  decrease  in  their 
degree  of  dissociation  (43);  hence  the  caseinate  is  precipitated 
in  combination  with  the  proportion  of  base  which  was  bound 
up  by  it  in  its  aqueous  solution,  because,  immediately  preceding 
precipitation,  the  combined  base  is  bound  up  in  an  undissociated 
molecule. 

It  has  also  been  shown  that  serum-globulin  is  precipitated 
from  serum  by  alcohol  in  the  form  of  a  salt  with  a  base,  and  not 
in  the  form  of  the  free  protein,  since  the  mixed  proteins  which 
may  thus  be  precipitated  are  completely  soluble  in  distilled  water, 
and  this  solution  yields  a  precipitate  on  passing  CO2  through  it 
(42). 

It  would  not  be  safe  to  assume,  however,  without  special  in- 
vestigation, that  alcohol  precipitates  all  protein  salts  without 
alteration  of  the  relative  proportion  of  base  or  acid  to  protein 
which  subsists  in  these  compounds  as  they  exist  in  aqueous 
solution.  So  far  this  can  only  be  positively  affirmed  for  the 
"neutral"  and  "basic"  casemates,  that  is,  the  caseinates  the 
solutions  of  which  are  respectively  neutral  to  litmus  and  to 
phenolphthalein. 

The  influence  of  other  coagulating  agents  upon  the  composition 
of  the  protein  salts  which  they  precipitate  is  even  more  uncertain 
than  that  of  alcohol,  and  in  one  case  at  all  events,  namely  that 
of  the  precipitation  of  sodium  caseinate  from  solutions  containing 
a  considerable  excess  of  sodium  hydrate  by  means  of  ammonium 
sulphate,  we  are  in  possession  of  definite  evidence  that  the  act 
of  coagulation  is  accompanied  by  a  change  in  the  composition 
of  the  protein  salts. 


70 


CHEMICAL  STATICS 


Spiro  and  Pemsel  (49)  employed  ammonium  sulphate  to  precipi- 
tate protein  salts;  their  procedure  was  as  follows:  A  given  weight 
of  protein  was  dissolved  in  a  measured  quantity  of  standardized 
NaOH  solution,  and  the  protein-base  compound  was  then  precipi- 
tated by  the  addition  of  a  suitable  excess  of  a  saturated  solution 
of  ammonium  sulphate.  The  quantity  of  alkali  bound  by  the 
protein  and  precipitated  with  it  was  then  determined  by  titration 
of  the  filtrate.  The  following  were  their  results,  employing  casein: 


Solution 

Mg  of  NaOH  pre- 
cipitated with  1 
gram  of  casein 

Equivalent  grm.  mol. 
of  NaOH  precipitated 
with  1  gram  casein 

1.14  grams  casein  in  10  cc.  N/5  NaOH.  . 
1.41  grams  casein  in  15  cc.  N/5  NaOH.  . 
1.24  grams  casein  in  20  cc.  N/5  NaOH.  . 
1.31  grams  casein  in  30  cc.  N/5  NaOH.  . 

26.1 
29.2 
33.1 
34.3 

65  X  10-6 
73  X  10~5 
82  X  10~5 
36  X  10~5 

Now  I  have  shown  (40)  employing  the  potentiometric  method 
(vide  infra),  which  is  a  static  measurement,  not  involving  any 
change  in  the  dynamical  condition  or  composition  of  the  body 
of  the  solution,  that  in  all  of  the  above  solutions  investigated  by 
Spiro  and  Pemsel  the  amount  of  alkali  which  was  actually  bound 
by  one  gram  of  casein  while  in  solution  must  have  been  at 
least  160  X  10~5  gram  equivalents.  Hence  it  is  clear  that  in 
the  act  of  coagulation  by  ammonium  sulphate  these  salts  of  casein 
are  very  materially  altered  in  composition. 

The  method  of  coagulation  by  heat  has  been  comparatively 
little  employed  for  the  investigation  of  protein  compounds  with 
acids.  It  cannot  be  employed  for  the  investigation  of  compounds 
of  proteins  with  bases  because  although  proteins  in  alkaline 
solution  are  denatured  by  heat  the  denatured  protein  is  only 
flocculated  in  an  acid  medium  (6).  It  has  been  shown  by  Chick 
and  Martin  that  in  acid  media  the  protein  coagulum  binds  a 
certain  proportion  of  the  acid,  but  whether  or  not  the  protein 
salt,  as  it  exists  in  the  solution  prior  to  coagulation,  is  flocculated 
without  alteration,  cannot  at  present  be  definitely  stated. 

At  the  present  stage  of  our  knowledge  therefore,  the  direct 
method  of  demonstrating  the  existence  of  protein  compounds 
cannot  be  trusted  to  yield  accurate  quantitative  data,  and  re- 
sults and  conclusions  based  upon  this  method  are  to  be  accepted 
with  the  greatest  caution. 


INDIRECT   METHOD  OF  PRECIPITATION  71 

3.  The  Direct  Method  of  Demonstrating  the  Existence  of 
Protein  Compounds  by  the   Solution  of  Otherwise  Insoluble 
Substances.  —  This   method    has    been    employed    by    W.    A. 
Osborne  (30)  who  has  shown  that  casein,  when  triturated  with 
calcium  carbonate,  carries  a  definite  proportion  of  the  calcium 
into  solution  in  the  form  of  the  soluble  calcium  caseinate  with 
the  evolution  of  COg.     Strychnin  is  similarly  carried  into  solu- 
tion by  casein. 

Osborne  and  Leavenworth  (31)  have  shown  that  edestin  will 
hold  in  solution  a  quantity  of  cupric  hydroxide  corresponding 
to  no  less  than  34.67  per  cent  of  its  weight  of  copper,  which, 
assuming  that  each  atom  of  copper  is  united  to  one  nitrogen 
atom,  implies  that  no  less  than  ten  out  of  every  sixteen  nitrogen 
atoms  contained  in  the  edestin  molecule  participate  in  forming 
the  compound  with  cupric  hydroxide.  This  is  exactly  the  pro- 
portion of  nitrogen  atoms  which  is  present  in  edestin  in  the 
form  of  —  COHN—  groups.  Precisely  similar  results  were  ob- 
tained with  gliadin. 

4.  The  Indirect  Method  of  Precipitation.  —  Cohnheim  and 
Krieger  (9)  have  elaborated  an  ingenious  method  of  determining 
the  acid-binding  capacity  of  the  proteins  which  is  based  upon 
the  fact  that  phosphotungstic  acid  forms  insoluble  salts  with 
proteins.     If  the  protein  is  combined  with  an  acid,  and  calcium 
phosphotungstate  is  employed,  double  decomposition  takes  place 
as  follows: 

Protein  hydrochloride  -f-  calcium  phosphotungstate 

(soluble)  (soluble) 

=  protein  phosphotungstate  -+-  CaC^ 

(insoluble) 

hence  the  quantity  of  calcium  chloride  present  in  the  filtrate, 
after  filtering  off  the  precipitate  and  carefully  washing  it  (34) 
is  taken  as  a  measure  of  the  quantity  of  hydrochloric  acid  which 
was  bound  by  the  protein  just  before  precipitation.  The  pro- 
cedure is  as  follows:  Weighed  amounts  of  protein  are  dissolved 
in  measured  volumes  of  acid  of  known  concentration.  The 
protein  is  then  precipitated  by  the  addition  of  an  excess  of  the 
phosphotungstate  and  the  filtrate,  plus  the  washings  of  the  pre- 
cipitate, is  titrated  against  standard  alkali.  An  excess  of  the 
phosphotungstate  is  stated  to  be  necessary  because  the  phospho- 
tungstate of  protein  is  believed  to  undergo  extensive  hydrolytic 


72 


CHEMICAL  STATICS 


dissociation  so  that  it  is  stable,  and  precipitation  is  complete, 
only  in  the  presence  of  an  excess  of  the  reagent. 

It  is  obvious  that  this  method  is  open  to  all  of  the  objections 
attaching  to  the  direct  method  of  precipitation  considered  above. 
In  our  present  stage  of  knowledge  we  are  very  much  in  the  dark 
concerning  the  actual  chemical  mechanism  of  the  precipitation 
of  proteins  by  reagents  such  as  phosphotungstic  acid  and  we  are 
by  no  means  certain,  indeed  it  is  highly  improbable,  that  only 
one  compound  of  a  given  protein  with  phosphotungstic  acid 
exists  or  that  its  composition  is  independent  of  the  excess  of 
phosphotungstic  acid  employed.  Hence  it  is  not  certain  that  in 
the  substitution  of  phosphotungstic  acid  for  hydrochloric  acid, 
under  the  conditions  outlined  above,  one  equivalent  of  phospho- 
tungstic acid  replaces  one  or  even  a  constant  number  of  equiva- 
lents of  hydrochloric  acid.  Results  obtained  by  this  method  are 
at  present,  therefore,  only  of  qualitative  value. 

The  experiments  of  Erb  (11),  who  employed  this  method, 
must  nevertheless  be  quoted  here  in  order  to  point  out  a  mis- 
conception which  his  interpretation  of  the  results  involves.  One 
cubic  centimetre  of  a  5  per  cent  watery  solution  of  vitellin  was 
mixed  with  1,  2,  3,  etc.,  cubic  centimetres  of  normal  HC1  and  the 
volume  of  each  solution  was  made  up  to  10  cc.  To  these  were 
added  a  constant  excess  of  calcium  phosphotungstate,  the  pre- 
cipitate was  filtered  off  and  washed,  and  the  filtrate  ti'trated. 
The  following  is  the  way  in  which  Erb  expresses  his  results : 


Cc.  of  normal 
HC1  added 

Cc.  AT/10  NaOH  re- 
quired to  neutral- 
ize the  filtrate 

HCl  bound 
in  cc.  AT/10 
HCl 

Theoretical 
excess  of  N/10 
HCl 

Hydrolytic 
dissociation 
per  cent 

1  gram 
protein  binds 
mg.  of  HCl 

0.1 

0.1 

0.9 

-1.9 

69.0 

63 

0.2 

0.8 

1.2 

-0.9 

59.6 

87 

0.3 

1.5 

1.5 

0.1 

48.3 

108 

0.5 

3.0 

2.0 

2.1 

31.0 

146 

0.7 

4.5 

2.5 

4.1 

13.7 

183 

0.9 

6.2 

2.8 

6.1 

3.4 

204 

1.0 

7.2 

2.8 

7.1 

3.4 

204 

1.5 

12.1 

2.9 

12.1 

0.0 

212 

It  will  be  seen  that  the -quantity  of  HCl  which  is  bound  by 
one  gram  of  vitellin  increases  with  increasing  concentration  of 
HCl  in  its  solution.  We  shall  see  that  this  phenomenon  is  a 
general  one  where  proteins  are  concerned.  It  appears  that  the 


INDIRECT   METHOD  OF  PRECIPITATION  73 

binding-capacity  of  the  vitellin  reaches  a  maximum  in  the  higher 
concentrations  of  acid.  Assuming  that  this  is  the  true  binding- 
capacity  of  vitellin  but  that  the  compound  which  is  formed  under- 
goes hydrolytic  dissociation  according  to  the  balanced  equation: 

Protein  hydrochloride  +  water  +±  Protein  +  HC1. 

Erb  calculates,  in  the  fourth  and  fifth  columns  of  the  above 
table,  respectively,  the  " theoretical  excess"  of  HC1  (unbound) 
and  the  percentage  hydrolytic  dissociation  of  the  protein  salt  in 
each  mixture.  This  assumption  is,  however,  unquestionably 
incorrect.  The  acid-  or  base-combining  capacity  of  the  proteins 
is  a  function  of  the  concentration  of  acid  or  base  in  their  solution 
and  not  independent  of  it,  as  it  is  in  the  case  of  a  strong  mono- 
basic acid  which  is  incapable  of  undergoing  polymerization.  This 
is  very  readily  shown  in  the  case  of  casein,  which  is  insoluble 
when  uncombined  and  which  would  therefore  be  precipitated  if 
its  salts  underwent  appreciable  hydrolytic  dissociation.  If  the 
alkali-binding  capacity  of  casein  were  independent  of  the  con- 
centration of  alkali  in  its  solution,  therefore,  the  addition  of  any 
quantity  of  alkali  beyond  that  necessary  to  carry  the  casein  into 
solution  should  be  without  effect  upon  the  number  of  equivalents 
of  alkali  neutralized  by  one  gram  of  casein.  As  one  increases 
the  amount  of  alkali  in  its  solution,  however,  the  alkali-binding 
capacity  of  casein,  as  estimated  by  the  potentiometric  method, 
increases  from  11.4  X  10~5  equivalents  per  gram  to  180  X  10~5 
equivalents  per  gram,  or  16  times  the  amount  of  alkali  neces- 
sary to  carry  it  into  solution  (40).  The  alkali-binding  capacity 
of  casein  is  therefore  not  a  constant  in  the  presence  of  varying 
amounts  of  alkali.  The  same  considerations  apply,  of  course, 
to  the  varying  combining-capacity  of  serum  globulin  in  the 
presence  of  a  varying  excess  of  acid  or  alkali  (39). 

In  the  case  of  proteins  which  are  soluble  in  water  when  un- 
combined with  acids  or  with  bases  the  demonstration  of  the  fact 
that  their  combining-capacities  vary  with  the  quantity  of  acid 
or  alkali  in  their  solutions  is  not  such  a  simple  matter,  but  it  is 
not  difficult  to  infer,  from  data  to  which  reference  will  be  made 
in  succeeding  chapters,  that  such  variations  in  combining-capac- 
ity are  an  essential  feature  of  the  behavior  of  these  proteins  also. 

It  is  obvious  that  almost  any  sufficiently  neutral  salt  of  an 
acid  which  forms  insoluble  compounds  with  the  proteins  might 


74 


CHEMICAL  STATICS 


be  employed  in  investigations  such  as  those  cited  above,  in  the 
place  of  calcium  phosphotungstate.  von  Rhorer  has  employed 
potassium  mercury  iodide  (K2HgI4)  and  calcium  picrate. 

5.  The  Method  of  Electrical  Conductivity.  —  This  method  is 
based  upon  the  fact  that  the  salts  of  the  proteins  are,  in  solution, 
less  highly  ionized  than  the  majority  of  the  strong  inorganic 
acids  or  bases  in  equivalent  concentration.  Hence  on  adding 
proteins  to  a  solution  of  one  of  these  latter,  the  total  number 
of  ions  per  liter  of  the  solution  is  diminished.  Moreover,  the 
protein  ions  which  are  formed  have  a  low  velocity  of  migration 
(16)  (37)  (38)  (40)  (41)  as  might  be  anticipated,  having  regard 
to  the  magnitude  of  their  mass  and  volume.  Hence,  on  adding 
protein  to  a  solution  of  a  strong  acid  or  of  a  strong  base,  the 
conductivity  of  the  solution  is  diminished  and  this  diminution 
affords  a  measure  of,  although  it  is  not  directly  proportional 
to,  the  quantity  of  acid  or  base  bound  by  the  protein.  This 
method  was  first  employed  in  a  systematic  manner  by  Sjoqvist 
(47) .  The  following  figures  are  illustrative  of  his  results : 


Egg  albumin  dissolved  in  0.025  N  HCl. 

Grams   albumin 
in  100  cc  
Mol.  Cond.  X107. 

0 

340.5 

0.53 
271 

1.06 
207 

1.59 
151 

2.13 
108 

2.55 

86 

3.19 
71.5 

4.25 
67 

5.23 

67 

6.38 
68 

The  molecular  conductivities  were  calculated  for  a  0.025  m. 
HCl  solution.  It  will  be  observed  that  after  a  certain  quantity 
of  albumin  has  been  dissolved  in  the  acid  (4.25  per  cent)  the 
molecular  conductivity  of  the  solution  approaches  constancy, 
indicating  that  all  of  the  acid  is  bound  by  the  protein;  the  infer- 
ence being  that  the  conductivity  thereafter  measured  is  that  of 
the  protein  hydrochloride  alone,  the  addition  of  further  albumin 
contributing  but  little  to  the  conductivity  of  the  solution,  since 
free,  unneutralized  protein  is  but  sparingly  ionized. 

Details  of  more  recent  results  obtained  by  this  method  and 
their  interpretation  will  be  found  in  the  chapters  dealing  with 
the  electrochemistry  of  the  proteins.  Since  this  measurement 
is  a  static  one,  the  objections  which  apply  to  the  two  methods 
previously  described  do  not  apply  to  it. 

6.  The  Cryoscopic  Method.  —  This  method  depends  upon  the 
diminution  in  the  total  ionic  +  molecular  concentration  in  solu- 


CRYOSCOPIC   METHOD  75 

tions  of  strong  acids  or  bases,  upon  the  addition  to  them  of  pro- 
teins. As  in  the  previous  method,  the  measurement  is  a  static 
one,  with  this  difference,  however,  that  the  measurement  is 
necessarily  conducted  at  or  in  the  neighborhood  of  0°  C.  and 
that  the  dynamical  equilibrium  of  the  system  under  investigation 
is  shifted  to  the  equilibrium  pertaining  at  this  temperature. 
Regarding  the  magnitude  of  this  shift  for  any  given  temperature 
range  we  have  but  meagre  data,  but  in  the  light  of  results  ob- 
tained with  the  caseinates  and  with  the  salts  of  ovomucoid  (Chap. 
IX)  it  is  probably  in  these  instances  and  for  the  temperature- 
range  30-0°  C.,  not  greater  than  the  experimental  error  of  the 
method. 

The  cryoscopic  method  was  first  employed,  for  this  purpose, 
by  Bugarzsky  and  Liebermann  (5),  who  found  that  upon  adding 
6.4  grams  of  egg  albumin  :to  100  cc.  of  0.05  N  HC1  or  NaOH 
the  difference  between  the  freezing-point  of  the  solution  and  that 
of  water  is  reduced  nearly  50  per  cent,  indicating  a  diminution 
by  nearly  50  per  cent  of  the  total  number  of  ions  plus  molecules 
per  liter  of  the  solution.  Upon  adding  similar  quantities  of 
protein  to  solutions  of  sodium  chloride,  little  or  no  alteration  in 
the  freezing-point  of  the  solutions  could  be  detected.  From 
these  results  it  is  usually  inferred  that  the  proteins  do  not  bind 
neutral  salts.  This,  however,  is  not  an  altogether  justifiable 
inference.  Hardy  (loc.  cit.)  has  detected  a  slight,  but  what  he 
considers  unmistakable,  depression  in  the  electrical  conductivities 
of  salt  solutions  upon  saturation  with  serum  globulin,  and  he 
assumes  that  a  compound  with  the  neutral  salt  is  actually  formed, 
but  that  it  is  only  stable  in  the  presence  of  excess  of  the  salt,  so 
that  at  any  given  salt-concentration  only  a  small  proportion  of 
the  salt  is  held  in  combination.  Mellanby  (28)  has  arrived  at 
similar  conclusions.  This  possibility  becomes  more  plausible 
when  we  view  it  in  the  light  of  the  Guldberg-Waage  mass  law. 
Representing  the  reaction  between  protein  and  NaCl  thus: 

Protein  +  NaCl  <=±  Protein— NaCl  Compound, 

we  may  suppose  that  the  station  of  equilibrium  lies  far  to  the 
right.  An  excess  of  NaCl  would  displace  this  equilibrium  towards 
the  left  and  a  greater  proportion  of  the  protein  would  exist  in 
the  solution  in  the  form  of  the  protein-salt  compound,  i.e.,  the 
compound  would  be  more  "stable."  Turning  now,  to  the  above- 


76 


CHEMICAL  STATICS 


quoted  results  of  Bugarzsky  and  Liebermann,  it  is  evident  that, 
save  in  the  presence  of  a  considerable  excess  of  neutral  salts, 
little  or  no  alteration  in  the  freezing-point  of  the  solution  would 
result  from  the  addition  to  it  of  protein,  since  the  depression  of 
conductivity  observed  by  Hardy  only  amounted  to  2  per  cent 
of  that  of  a  N/ 10  solution,  and  the  cryoscopic  method  is  not 
sufficiently  sensitive  to  reveal,  with  certainty,  such  slight  varia- 
tions in  the  freezing-point  of  solutions.  For  this  purpose  the 
method  of  electrical  conductivity  is  much  to  be  preferred.  An 
alternative  interpretation  of  these  results  will  be  found  in  Chap.  VI. 

7.  The  Potentiometric  Method.  —  This  method  is  also  based 
upon  the  fact  that  the  addition  of  protein  to  a  solution  of  a  strong 
base  or  acid  diminishes  the  concentrations  of  the  ions  of  the 
inorganic  constituent  of  the  mixture.  As  in  the  previous  methods, 
the  measurement  is  static.  But,  instead  of  involving  the  meas- 
urement of  the  total  ionic  or  ionic  plus  molecular  concentration 
of  the  mixture  this  method  enables  us  to  directly  determine  the 
concentration  of  a  given  ion.  The  results  are  therefore  much 
more  readily  interpreted  than  those  obtained  by  the  two  methods 
previously  described  and,  indeed,  furnish  us  with  a  direct  meas- 
ure of  the  quantity  of  a  (highly  dissociated)  base  or  acid  which 
is  actually  bound  in  the  solution,  by  the  protein.  For  a  brief 
description  of  the  principles  underlying  potentiometric  measure- 
ments in  concentration-chains  the  reader  is  referred  to  the  appen- 
dix; for  fuller  details  to  such  works  as  those  of  Hamburger  (15) 
and  Michaelis  (29).* 

The  potentiometric  method  was  first  employed  for  this  pur- 
pose by  Bugarzsky  and  Liebermann  (5).  These  investigators 
employed  two  different  concentration-chains.  The  one,  the 
mercury-chain,  was  built  up  as  follows: 


Hg 

HgCl  (solid),  HC1 

NaCl 

NaBr,  HgBr  (solid) 

Hg 

1 

2 

3 

4 

5 

In  the  first  instance  the  potential  of  the  chain  for  a  certain  con- 
centration of  HCi  in  2  was  measured,  then  a  weighed  amount 

*  For  the  modifications  in  technique  imposed  by  the  presence  of  proteins 
in  the  solutions  under  investigation  vide  Appendix. 


POTENTIOMETRIC  METHOD 


77 


of  protein  was  introduced  into  2  and  the  potential  again  deter- 
mined. The  difference  between  the  two  readings,  in  accordance 
with  the  Nernst  formula,  affords  a  measure  of  the  number  of 
Cl  ions  bound  by  the  protein.  The  other  concentration-cell 
was  the  ordinary  gas-chain. 


Pt  saturated  with  H2 

Acid 

Alkali 

Pt  saturated  with  H2 

1 

2 

3 

4 

As  before,  the  potential  of  the  chain  was  first  measured  for  a 
known  concentration  of  acid  in  2,  then  a  weighed  amount  of 
protein  was  added  to  the  acid  and  the  potential  of  the  chain  was 
again  determined,  the  difference  between  the  two  readings  afford- 
ing, in  this  case,  a  measure  of  the  diminution  in  the  number  of 
H  ions  due  to  the  introduction  of  the  protein.  When  it  is  desired 
to  determine  the  base-binding  capacity  of  the  protein,  the  latter 
is  added  to  the  alkali  in  3,  instead  of  to  the  acid  in  2. 

The  following  are  representative  of  the  results  obtained  by 
Bugarzsky  and  Liebermann.  In  the  tables  G  signifies  the  number 
of  grams  of  protein  in  "100  cc.  of  solution,  p  the  percentage  of 
acid  or  base  bound  by  the  protein,  and  r  the  ratio. 

grams  of  acid  or  base  bound 
grams  of  protein 

I.    GAS-CHAIN.    EGG  ALBUMIN  DISSOLVED  IN  0.05  N  HCI 


G 

p 

r 

G 

P 

r 

0 

0.4 
0.8 
1.6 

0 
9.0 
18.9 
33.3 

0'042" 
0.044 
0.038 

3.2 
6.4 
12.8 

60.2 
96.6 
99.7 

0.034 
0.027 
0.014 

II.    MERCURY-CHAIN.    EGG   ALBUMIN  DISSOLVED  IN  0.05  N  HCI 


G 

p 

r 

G 

p 

r 

0 

0 

1.6 

38.0 

0.044 

0.4 

10.7 

0.048 

3.2 

64.0 

0.037 

0.8 

20.2 

0.046 

6.4 

76.0 

0.022 

78 


CHEMICAL  STATICS 


III.    GAS-CHAIN.    EGG  ALBUMIN   DISSOLVED  IN  0.05  N  NaOH 


G 

p 

r 

G 

p 

r 

• 

0 

0 

3.2 

60.2 

0.037 

0.8 

14.4 

0.035 

6.4 

97.0 

0.030 

1.6 

27.4 

0.034 

12.8 

99.9 

0.016 

It  will  be  seen  that  as  the  concentration  of  protein  rises,  i.e., 
as  the  proportion  of  base  or  acid  to  protein  diminishes,  the  base- 
or  acid-binding  capacity  of  the  protein  diminishes.  We  have 
already  had  occasion  to  note  this  fact  in  connection  with  the 
results  of  Erb  (11). 

Employing  a  mercury  chain  containing  the  element  (HgCl 
(solid),  NaCl)  instead  of  the  element  (HgCl  (solid),  HC1)  used 
above,  and  adding  to  the  NaCl  solution  in  this  element  varying 
quantities  of  albumin,  the  following  results  were  obtained: 

IV.    MERCURY-CHAIN.    EGG  ALBUMIN  DISSOLVED  IN  0.05  N  NaCl 


G 

E  (electromotive  force 
of  chain) 

G 

E  (electromotive  force 
of  chain) 

0 

0.4 
0.8 

0.1287 
0.1280 
0.1279 

1.6 
3.2 
6.4 

0.1278 
0.1281 
0.1275 

It  will  be  seen  that  the  electromotive  force  of  the  chain  is  but 
little  affected  by  the  addition  of  egg  albumin  to  the  element 
containing  NaCl. 

The  method  employed  by  Bugarzsky  and  Liebermann  involved, 
as  we  have  seen,  a  direct  comparison  of  the  ionic  concentrations 
in  the  protein  solution  and  in  the  solvent,  respectively.  This 
comparison  may  be  avoided  and  the  existence  of  a  variety  of 
protein  compounds  inferred  from  the  changes  in  curvature  of 
the  curve  of  ionic  concentration  consequent  upon  the  admixture 
with  the  protein  solution  of  varying  proportions  of  the  substance 
with  which  compounds  are  formed.  This  method,  which  is 
essentially  the  same  as  that  now  widely  employed  in  various 
branches  of  analytical  chemistry  (3)  (46)  (18)  (19),  was  first 
applied  to  protein  solutions  by  D'Agostino  and  Quagliariello 
(10)  and  has  recently  been  extensively  employed  by  C.  L.  A. 


METHOD  OF  CATALYSIS  79 

Schmidt  (45)  who,  with  its  aid,  has  been  able  to  sharply  dis- 
tinguish between  the  precipitation  of  a  protein  through  the 
formation  of  a  true,  insoluble  compound,  and  flocculation  of  the 
protein  through  alteration  of  its  physical  condition  or  degree 
of  hydration;  the  so-called  " salting-out"  effect. 

The  details  of  recent  investigations  by  the  potentiometric 
method  and  their  interpretation  will  be  found  in  the  chapters 
dealing  with  the  electrochemistry  of  the  proteins. 

8.  The  Method  of  Catalysis.  —  In  this  method,  as  in  the 
above,  direct  measurements  of  the  number  of  hydrogen  or 
hydroxyl  ions  bound  by  the  protein  are  secured.  The  measure- 
ment is  not,  however,  altogether  a  static  one,  since  a  foreign 
substance  is  added  to  the  solution  of  the  protein,  and  the  rate 
of  change  of  this  substance  is  the  quantity  actually  measured. 
When  the  change  in  this  substance  leads  to  the  setting  free  of 
an  acid  (as,  for  example,  in  the  catalysis  of  methyl  acetate)  the 
method  is  open  to  serious  objection. 

As  is  well  known,  many  reactions  are  accelerated  by  hydroxyl 
and  hydrogen  ions,  and  the  velocity-constants  of  these  reactions 
(such  as  the  inversion  of  sugar  by  acids,  the  saponification  of 
methyl  acetate  by  alkalies,  etc.)  are  directly  proportional  to  the 
concentration  of  the  hydroxyl  or  hydrogen  ions  in  the  reacting 
system.  If,  therefore,  we  act  upon  such  a  mixture  with  a  given 
concentration  of  an  acid,  or  of  a  base,  as  the  case  may  be,  and 
observe  the  velocity  of  the  transformation,  and,  if  we  then  act 
upon  a  similar  mixture  with  the  same  quantity  of  acid,  to 
which,  however,  a  weighed  amount  of  protein  has  been  added, 
and  again  determine  the  velocity  of  the  transformation,  the 
difference  between  the  two  velocities  affords  a  measure  of  the 
numbers  of  H  ions  (or  of  OH  ions,  as  the  case  may  be)  which 
have  been  bound  through  the  introduction  of  the  protein.  This 
method  was  employed  by  Cohnheim  (8)  who  states  that  it  was 
first  used  for  this  purpose  by  Hoffman,  at  the  suggestion  of 
Wilh.  Ostwald. 

As  a  reaction  which  is  accelerated  by  H+  ions  Cohnheun  em- 
ployed the  inversion  of  cane  sugar.  This  reaction,  in  the  presence 
of  free  acid,  obeys  the  monomolecular  formula 


80  CHEMICAL  STATICS 

where  A  is  the  initial  concentration  of  the  cane-sugar,  X  is  the 
amount  inverted  after  a  time  t,  and  k  is  a  constant  (at  constant 
temperature)  which  varies  directly  as  the  hydrion  concentration 
of  the  solution.  The  period  during  which  the  reaction  proceeded 
was  constant  (4  hours) ;  hence,  if  X  be  the  amount  of  sugar  trans- 
formed after  the  addition  to  its  solution  of  a  measured  volume 
of  HCl-solution,  and  X'  that  after  the  addition  to  the  same  con- 
centration of  cane-sugar  of  the  same  volume  of  the  HCl-solution, 
to  which,  however,  protein  has  been  added  then: 

C       log  A -log  (A -30 
C'      logA-log(A-JT)' 

where  C  is  the  concentration  of  hydrions  in  the  pure  acid,  and 
C"  that  in  the  acid  to  which  protein  has  been  added.  Knowing 
the  value  of  C  that  of  C"  can  be  immediately  calculated.  From 
his  results  Cohnheim  concluded  that  0.25  gram  of  protalbu- 
mose,  dissolved  in  5  cc.  of  solution  containing  0.025  gram  of 
HC1,  combines  with  4.16  per  cent  of  its  own  weight  of  the  acid, 
—  similar  determinations  were  made  with  other  albumoses. 

Hardy  (16)  has  employed  the  catalysis  of  the  inversion  of  cane 
sugar  by  H+  ions  in  the  measurement  of  the  acid-binding  capacity 
of  serum-globulin,  and  that  of  the  saponification  of  methyl  acetate 
by  OH'  ions  in  the  measurement  of  the  base-combining  capacity 
of  this  protein. 

In  this  connection  (although,  owing  to  the  complexity  of  the 
experimental  conditions,  they  have,  as  yet,  but  a  qualitative 
significance)  the  results  obtained  by  a  number  of  observers  (12) 
(7)  (22)  (23)  (2)  (1)  (53)  (35)  on  the  protection  conferred  by 
proteins  against  the  destruction  of  enzymes  by  acid  or  alkali  may 
be  mentioned.  As  is  well  known,  the  proteolytic  enzymes,  for 
example,  are  very  rapidly  destroyed  by  an  excess  of  free  acid 
or  base,  the  action  of  the  acid  or  base  being,  probably,  that  of 
catalysing  the  destruction  (hydrolysis?)  of  the  enzyme  which 
occurs  even  in  pure  water.  If,  however,  protein  be  added  to 
acid  or  alkaline  solutions  containing  these  enzymes,  the  rate  of 
destruction  of  the  enzyme  is  greatly  diminished,  and  this  has 
been  attributed  by  Falk  (12),  Langley  and  Edkins  (22),  Langley 
and  Eves  (23)  and  Vernon  (53)  to  a  binding  of  the  free  acid  or 
alkali  by  the  added  protein.  It  must  not  be  forgotten,  however, 
that  the  proteolytic  enzymes  are,  in  the  presence  of  protein, 


INDICATOR  METHOD  81 

probably  combined  in  some  proportion  with  the  protein,  which 
may  thus  serve  in  a  double  manner  to  protect  them  from  de- 
struction. This  fact,  however,  only  adds  to  the  complexity  of 
the  conditions,  without  militating  against  the  correctness  of  the 
view  urged  by  the  investigators  quoted  above  that  the  protein 
protects  the  enzyme  by  binding  some  of  the  excess  of  acid  or  base. 

9.  The  Indicator  Method.  —  This  is  simply  the  ordinary 
method  of  acidimetry  or  alkalimetry  applied  to  solutions  contain- 
ing protein,  and  has  been  very  extensively  used;  unfortunately, 
in  the  past,  without  a  very  clear  understanding  of  the  exact  sig- 
nificance of  the  data  obtained.  In  order  to  appreciate  this  fact 
it  is  necessary  to  recollect  that  the  method,  as  ordinarily  applied, 
is  not  a  static  one.  Not  only  is  a  foreign  substance,  namely 
the  indicator,  added  to  the  system  under  investigation,  but  while 
acid  or  alkali  is  being  added  to  the  system  to  secure  neutrality 
to  the  chosen  indicator,  the  combining  capacity  of  the  protein 
is  continually  changing  in  response  to  the  altering  reaction  of  its 
solution.  The  final  result  obtained  in  this  manner  with  a  given 
indicator  tells  us  nothing  save  the  condition  of  equilibrium  in 
the  solution  at  the  precise  H+  or  OH'  concentration  at  which  that 
indicator  changes  color;  it  cannot  yield  us  information  concern- 
ing the  acid  or  alkali  binding  capacity  of  the  protein  at  any  other 
H+  or  OH'  concentration.  In  the  further  application  of  this 
method,  the  methods  of  acidimetry  and  alkalimetry  devised  by 
Friedenthal  and  Salm  (14)  (13)  (44)  and  elaborated  by  Sorensen 
(48)  are  indubitably  destined  to  prove  of  the  greatest  utility,  and 
arouse  the  hope  that  the  indicator  method  may  be  of  more  service 
to  the  protein  chemist  in  the  future  than  it  has  been  in  the  past. 

The  use  of  indicators  in  protein  solutions  is,  however,  accom- 
panied by  two  notable  drawbacks.  In  the  first  place,  owing  to 
the  amphoteric  character  of  the  proteins  and  also  to  their  mul- 
tiple combining  capacities,  the  changes  in  the  hydrogen-  or 
hydroxyl-ion  content  of  protein  solutions,  upon  the  addition  of 
acid  or  of  alkali,  over  a  considerable  range,  are  relatively  slight, 
and,  for  this  reason,  sharp  end-reactions  are  rarely  obtained  with 
indicators  in  protein  solutions,  unless  their  color  changes  occur  at 
H+  or  OH'  concentrations  lying  without  or  upon  the  boundaries  of 
this  range.  Then,  again,  many  of  the  substances  commonly  used 
as  indicators  in  acidimetry  and  alkalimetry  combine  chemically 
with  the  proteins,  and  the  compounds  thus  formed  are  not  in- 


82  CHEMICAL  STATICS 

frequently  either  insoluble  or  of  a  different  color  from  the  free 
indicator  or  its  combination  with  inorganic  acids  or  bases  (27) 
(17)  (36). 

10.  The  Method  of  "Masking"  the  Physiological  Effects  of 
Ions  by  the  Addition  of  Proteins  to  their  Solutions.  —  This 
method  although,  as  yet,  only  of  qualitative  importance,  is 
nevertheless  of  surpassing  interest  to  the  physiologist,  since  the 
fluids  which  bathe  the  tissues  contain  notable  quantities  of 
protein,  which  may  be  supposed  to  modify,  in  a  greater  or  less 
degree,  the  physiological  action  of  the  inorganic  substances 
which  they  contain.  This  method  appears  to  have  first  been 
employed  by  Loeb  (24),  and  it  has  since  been  utilized  by  Stiles 
and  Beers  (50)  who,  among  other  observations,  have  shown  that 
the  onset  of  rigor,  which  rapidly  occurs  when  frogs'  muscles  are 
immersed  in  solutions  of  barium  salts,  is  greatly  delayed  when 
protein  is  added  to  the  solutions.  These  experiments  are,  how- 
ever, not  conclusive  since  they  are  open  to  the  criticism  that  the 
added  protein  may  alter  the  permeability  of  the  tissue  for  inor- 
ganic ions,  for  example,  by  clogging  up  the  pores  of  the  external 
limiting  membranes.  This  criticism,  although  a  serious  one  from 
the  chemical  standpoint,  does  not,  however,  detract  from  the 
interest  to  physiologists  of  such  experiments,  since,  whatever  the 
mechanism  may  be  which  leads  to  modification  by  proteins  of 
the  physiological  effects  of  inorganic  substances  in  solution,  the 
probable  importance  of  such  effects  in  life-phenomena  is  the 
same. 

La  Franca  (21)  has  published  the  results  of  a  series  of  experi- 
ments which,  in  the  light  of  the  important  investigations  of 
Madsen  and  Nyman  (26)  hold  out  the  hope  that  this  method 
may  ultimately  be  employed  in  a  quantitative  manner.  This 
observer,  employing  the  method  of  Paul  and  Kronig  (20)  (32), 
determined  the  toxicity,  for  bacteria,  of  solutions  of  copper 
sulphate,  mercurous  nitrate  and  silver  nitrate  to  which  varying 
amounts  of  protein  had  been  added;  at  the  same  time  he  meas- 
ured the  concentration  of  heavy-metal  ions  in  the  solution  by 
the  potentiometric  method.  His  results  show  a  satisfactory 
parallelism  between  the  diminution  in  the  toxicities  of  these 
solutions  caused  by  the  addition  of  the  protein,  and  the  number 
of  heavy-metal  ions  bound  by  the  protein,  as  revealed  by  the 
potentiometric  measurements. 


LITERATURE  CITED 


LITERATURE   CITED 

(1)  Bayliss,  W.  M.,  and  Starling,  E.  H.,  Journ.  of  Physiol.  30  (1903),  p.  61. 

(2)  Biernacke,  E.,  Zeit.  f.  Biol.  28  (1891),  p.  49. 

(3)  Bottger,  W.,  Zeit.  f.  Physik.  Chem.  24  (1897),  p.  253. 

(4)  Bredig,  G.,  Zeit.  f.  Elektrochem.  6  (1899),  p.  33. 

(5)  Bugarszky,  S.,  and  Liebermann,  L.,  Arch.  f.  d.  ges.  Physiol.  72  (1898), 

p.  51. 

(6)  Chick,  H.,  and  Martin,  C.  J.,  Journ.  Physiol.  45  (19i2),  pp.  61  and  261. 

(7)  Chittenden,  R.  H.,  and  Ely,  J.  S.,  Amer.  Chem.  Journ.  4  (1882),  p.  107. 

(8)  Cohnheim,  O.,  Zeit.  f.  Biol.  33  (1896),  p.  489. 

(9)  Cohnheim,  O.,  and  Krieger,  H.,  Zeit.  f.  Biol.  40  (1900),  p.  95. 

(10)  D'Agostino,  E.,  and  Quagliariello,  G.,  Nernst  Festschrift.  (1912),  p.  27. 

(11)  Erb,  W.,  Zeit.  f.  Biol.  41  (1910),  p.  309. 

(12)  Falk,  F.,  Virchow's  Arch.  84  (1881),  p.  119. 

(13)  Fels,  B.,  Zeit.  f.  Elektrochem.  10  (1904),  p.  208. 

(14)  Friedenthal,  H.,  Zeit.  f.  Allgem.  Physiol.  4  (1904),  p.  44;  Zeit.  f.  Elek- 

trochem. 10  (1904),  p.  113. 

(15)  Hamburger,  H.  J.,  "Osmotischer  Druck  und  lonenlehre."    Wiesbaden, 

1907. 

(16)  Hardy,  W.  B.,  Journ.  of  Physiol.  33  (1905),  p.  251. 

(17)  Heidenhain,  M.,  Arch.  f.  d.  ges.  Physiol.  90  (1902),  p.  115. 

(18)  Hildebrand,  J.  H.,  Journ.  Amer.  Chem.  Soc.  35  (1913),  p.  847. 

(19)  Hildebrand,  J.  H.,  and  Bowers,  W.  G.,  Journ.  Amer.  Chem.  Soc.  38 

(1916),  p.  785. 

(20)  Kronig,  B.,  and  Paul,  Th.,  Zeit.  f.  Hyg.  und  Infekt.  Krankheiten  25 

(1897),  p.  1. 

(21)  La  Franca,  S.,  Zeit.  f.  physiol.  Chem.  48  (1906),  p.  481. 

(22)  Langley,  J.  N.,  and  Edkins,  J.  S.,  Journ.  of  Physiol.  7  (1886),  p.  371. 

(23)  Langley,  J.  N.,  and  Eves,  F.,  Journ.  of  Physiol.  4  (1883),  p.  18. 

(24)  Loeb,  J.,  Amer.  Journ.  of  Physiol.  3  (1900),  p.  327. 

(25)  LundSn,  H.,  Journ.  Biol.  Chem.  4  (1908),  p.  267. 

(26)  Madsen,  T.,  and  Nyman,  M.,  Zeit.  f.  Hyg.  und  Infekt.  Krankheiten.  57 

(1907),  p.  388;    Communication  de  1'Inst.  Serotherap.  de  PEtat 
Danois  2  (1908). 

(27)  Mathews,  A.  P.,  Amer.  Journ.  Physiol.  1  (1898),  p.  445. 

(28)  Mellanby,  J.,  Journ.  of  Physiol.  33  (1905),  p.  338. 

(29)  Michaelis,  L.,  "Die  Wasserstoffionen  konzentration,"  Berlin  (1914). 

(30)  Osborne,  W.  A.,  Journ.  of  Physiol.  27  (1901),  p.  398. 

(31)  Osborne,  T.  B.,  and  Leavenworth,  C.  S.,  Journ.  Biol.  Chem.  28  (1916), 

p.  109. 

(32)  Paul,  T.,  and  Kronig,  B.  Zeit.  f.  physik.  Chem.  21  (1896),  p.  421. 

(33)  Platner,  A.  E.,  Zeit.  f.  Biol.  2  (1866),  p.  417. 

(34)  Von  Rhorer,  L.,  Arch.  f.  d.  ges.  Physiol.  90  (1902),  p.  368. 

(35)  Rosenthaler,  L.,  Biochem.  Zeit.  26  (1910),  p.  9. 

(36)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  4  (1904),  p.  1. 

(37)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  11  (1907),  pp.  437 

and  542. 


84  CHEMICAL  STATICS 

(38)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  12  (1908),  p.  473. 

(39)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  5  (1908),  p.  155. 

(40)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  14  (1910),  p.  528. 

(41)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  14  (1910),  p.  601. 

(42)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  7  (1910),  p.  351. 

(43)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  15  (1911),  p.  387. 

(44)  Salm,  E.,  Zeit.  f.  Elektrochem.  10  (1904),  p.  341;    Zeit.  f.  physikal 

Chem.  57  (1905),  p.  471.,  cf.  also  Clark,  W.  M.,  and  Lubs,  H.  A., 
Journ.  Bact.  2  (1917),  p.  1. 

(45)  Schmidt,  C.  L.  A.,  Journ.  Biol.  Chem.  25  (1916),  p.  63.    Univ.  of 

Calif.  Publ.  Pathol.  2  (1916),  p.  157. 

(46)  Schmidt,  C.  L.  A.,  and  Finger,  C.  P.,  Journ.  Physical  Chem.  12  (1908), 

p.  406. 

(47)  Sjoqvist,  J.,  Skand.  Arch.  f.  Physiol.  5  (1895),  p.  277. 

(48)  Sorensen,  S.  P.  L.,  Ergeb.  d.  Physiol.  12  (1912),  p.  393. 

(49)  Spiro,  K.,  and  Pemsel,  W.,  Zeit.  f.  physiol.  Chem.  26  (1898),  p.  233. 

(50)  Stiles,  P.  G.,  and  Beers,  W.  H.,  Amer.  Journ.  Physiol.  14  (1905),  p.  133. 

(51)  Strecker,  A.,  Annalen  der  Chem.  148  (1868),  p.  87. 

(52)  Van  Slyke,  L.  L.,  and  Hart,  E.  B.,  Amer.  Chem.  Journ.  33  (1905),  p.  461 . 

(53)  Vernon,  H.  M.,  Journ.  of  Physiol.  31  (1904),  p.  346. 


CHAPTER  V 
THE   COMPOUNDS   OF  THE   PROTEINS    (Continued) 

1.  Stoichiometrical  Relations  in  Protein  Compounds.  —  With 
the  aid  of  the  various  methods  outlined  in  the  previous  chapter,  the 
existence  of  a  number  of  compounds  of  the  proteins  with  inorganic 
acids,  bases  and  salts  has  been  conclusively  demonstrated.  Of 
these  compounds,  however,  comparatively  few  have  been  exten- 
sively studied,  and  in  equally  few  cases  have  the  Stoichiometrical 
relations  which  pertain  between  the  constituents  of  the  compounds 
been  elucidated.  This  is  due  in  part,  no  doubt,  to  the  complexity 
and  amphoteric  character  of  the  proteins  themselves,  and  in  part 
to  the  difficulty  of  sharply  characterizing  and  of  isolating  the 
individual  proteins  in  a  pure  condition,  but  in  the  main,  I  believe, 
to  the  fact  that  physical  chemistry  has  only  recently  been  in  a 
position  to  supply  us  with  the  implements  which  the  investigation 
of  these  compounds  demands.  We  have  seen  that  the  successful 
investigation  of  these  compounds  requires,  as  a  rule,  the  employ- 
ment of  static  methods  of  measurement;  methods,  that  is,  which 
do  not  involve  a  disturbance  of  the  equilibria  in  the  system  while 
these  equilibria  are  being  determined.  Many  investigators  in  this 
field  have,  in  the  past,  employed  methods  of  determining  the  exist- 
ence of  protein  compounds,  which,  to  our  modern  perceptions, 
clearly  involved  a  variable  and  uncontrolled  interference  with  the 
very  equilibrium  which  was  the  subject  of  investigation.  Exam- 
ination of  more  modern  literature,  however,  cannot  fail  to  impress 
the  reader  with  the  conviction  that  in  proportion  to  the  adequacy 
of  the  chemical  or  physico-chemical  technique  employed,  Stoi- 
chiometrical relations  between  the  proteins  and  the  substances  with 
which  they  combine  are  revealed  or  indicated. 

The  problem  of  determining  the  nature  of  the  compounds 
formed  by  the  proteins  with  inorganic  acids  or  bases  is  complicated 
by  the  fact  that,  as  a  rule,  a  protein  can  combine  with  not  only  one 
but  several  equivalents  of  a  base  or  acid,  so  that  on  adding  acid  or 
alkali  to  a  solution  of  a  protein  we  obtain  a  continuously  varying 
mixture  of  the  various  possible  salts.  Precisely  analogous  phenom- 

85 


86  CHEMICAL  STATICS 

ena  are,  however,  met  with  among  the  complex  and  double  salts, 
and  the  salts  of  polybasic  acids  or  poiyacid  bases  in  the  domain  of 
inorganic  chemistry,  and  they  afford  no  legitimate  grounds  for  the 
belief  that  the  protein  compounds  are  not  molecular  in  character 
and  consequently  do  not  obey  the  law  of  constant  combining  pro- 
portions. All  of  the  accurate  data  which  we  possess  lend  decided 
support  to  the  view  that  although  the  proteins  may  enter  into 
combination  in  multiple  proportions,  these  proportions  are  con- 
stant and  indicate  that  under  definite  conditions  of  reaction  and 
concentration  the  protein  molecules  possess  a  definite  and  measur- 
able equivalent  weight. 

For  the  investigation  of  such  compounds  exactly  the  type  of 
technique  must  be  employed  which  has  proved  to  be  successful  in 
dealing  with  the  inorganic  compounds  of  analogous  complexity, 
modified,  however,  by  the  limitations  imposed  upon  us  by  the 
instability  of  the  proteins  themselves.  From  the  previous  chapter 
it  will  be  clear  that  in  the  majority  of  instances  the  characteriza- 
tion of  the  protein  compounds  must,  for  the  present  and  pending 
further  elaboration  of  our  technique,  be  based  upon  electrochemical 
measurements.  A  discussion  of  the  data  derived  from  measure- 
ments of  this  type  must  necessarily  be  deferred  until  later  chapters 
in  which  the  electrochemical  behavior  of  the  protein  salts  will  be 
taken  up  in  detail.  For  the  present,  therefore,  except  in  the  case 
of  the  protamins,  of  which  the  salts  with  acids  lend  themselves  to 
sharp  characterization  by  ordinary  chemical  methods,  we  shall 
simply  dwell  upon  the  existence  and  general  properties  of  certain 
protein  compounds  without  seeking  to  decide,  in  any  specific 
instance,  how  many  equivalents  of  the  protein  or  of  the  remaining 
constituent  are  bound  up  in  one  molecule  of  the  salt. 

2.  The  Compounds  of  the  Protamins  with  Inorganic  Acids 
and  Bases.  —  The  protamins  occur  combined  with  nucleic 
acid,  in  the  nuclei  of  spermatozoa  (27)  (28)  (13).  According  to 
Burian  (10),  the  nucleo-protein  in  the  spermatozoa  of  the  salmon 
is  formed  by  the  combination  of  one  molecule  of  nucleic  acid  with 
one  molecule  of  salmin. 

The  protamins  (19)  (20)  are  predominantly  basic  substances, 
the  acid  function  being  very  small  in  comparison.  They  are 
soluble  in  water,  yielding  strongly  alkaline  solutions.  The  formula 
of  salmin  sulphate  is  a  multiple  of: 

C3oH67N1706,  2  H2S04  +  H20 


PROTAMINS  87 

while  that  of  clupein  sulphate  is  a  multiple  of 
C3oH57N1706,  2  H2SO4; 

but  if  an  insufficient  quantity  of  acid  is  present  to  completely 
neutralize  the  protamin,  "basic"  salts,  containing  fewer  equiva- 
lents of  acid,  are  readily  formed. 

The  sulphuric  acid  in  these  compounds  is  readily  replaced  by  an 
equivalent  amount  of  hydrochloric  (13),  nitric,  carbonic,  or 
chromic  acids.  The  nitrates,  chlorides  and  carbonates  are  readily 
soluble,  the  sulphates  sparingly  soluble  in  cold,  but  more  soluble 
in  hot,  water,  while  the  chromates  are  insoluble.  The  chloride  of 
salmin  is  soluble  in  methyl  alcohol  (13). 

The  formula  of  sturin  sulphate  is,  according  to  Kossel,  a  multiple, 
either  of 

2C33H61N1707 

or,  more  probably,  of 

4C36H69N1907 

Scombrin  (derived  from  mackerel)  sulphate  possesses,  according 
to  Kurajeff  (21),  the  formula: 

C3oH6oN1606,2H2S04. 

It  is  soluble  in  water.  The  chromate  is  insoluble  in  water  and 
possesses  the  formula: 

CsoHgsNieOs,  2  H2Cr04, 

differing,  therefore,  from  the  sulphate,  not  only  in  the  acid  com- 
ponent, but  also  in  the  absence  of  one  molecule  of  water  for  every 
two  molecules  of  combined  acid.  It  is  to  be  particularly  noted 
that  the  less  soluble  protein  salt  contains  less  water  combined  with  the 
protein.  Remarkably  analogous  to  this  is  the  fact  that  whereas 
salmin  sulphate  is  freely  diffusible  through  parchment  paper  (53), 
clupein  sulphate,  which  contains  one  molecule  less  of  water  for 
every  two  molecules  of  combined  acid,  is  almost  indiffusible  (49). 
We  shall  have  occasion  to  refer  to  analogous  facts  when  considering 
the  mechanism  of  the  coagulation  of  proteins  (Chap.  XII). 

The  protamins  also  form  compounds  with  chloroplatinic  acid  (28) ; 
these  compounds  are  insoluble  in  neutral  water,  alcohol  or  ether, 
but  are  soluble  in  water  containing  an  excess  of  acid.  According 
to  Goto  (13),  the  formula  of  the  chloroplatinate  of  salmin  is: 

C3oH57N1706,  4  HC1,  2  PtCl4. 


88  CHEMICAL  STATICS 

Although  the  acid  function  of  the  protamins  is  very  weak  in 
comparison  with  the  basic  function,  salts  with  inorganic  bases  are 
nevertheless  formed.  The  salts  of  the  heavy  metals  (except  cupric 
salts)  produce  from  alkaline  solutions  of  free  protamin  precipi- 
tates of  the  corresponding  salts  of  the  protein.  The  compound 
with  divalent  copper  is  soluble  in  water,  and  its  solutions  are 
violet  in  color.  The  compounds  with  monovalent  copper  are  very 
sparingly  soluble.  On  adding  alkali  (sodium  hydrate)  in  excess 
of  one-half  the  equivalent  of  the  protein  (clupein)  molecule,  to  the 
precipitate  of  the  silver  salt,  a  yellow  color  is  produced,  indicating 
that  sodium  has  displaced  some  of  the  silver  from  the  molecule  and 
that  the  silver  protaminate  is  now  mixed  with  silver  oxide. 

Other  compounds  which  the  protamins  form  will  be  considered 
in  the  succeeding  chapter.  The  physical  properties  of  the  pro- 
tamins and  their  salts  will  be  discussed  in  the  chapters  dealing  with 
the  physical  properties  of  protein  systems. 

3.  The  Compounds  of  Casein  with  Bases,  Acids,  and  Salts.  — 
Casein,  in  the  dry  state,  forms  a  white  powder,  the  general  proper- 
ties of  which  have  been  described  in  Chap.  II.  Uncombined  and 
purified  casein  is  insoluble  in  distilled  water  or  in  very  dilute  salt 
solutions  (11)  (34)  (22)  (36).  A  suspension  of  casein,  thoroughly 
shaken  up  in  distilled  water,  reddens  litmus  paper  wherever  the 
suspended  particles  touch  it,  but  if  this  suspension  be  filtered  the 
filtrate  contains  no  detectable  casein  and  is  neutral  to  litmus. 
Casein  is  therefore  probably  soluble  to  a  very  slight  extent  in  dis- 
tilled water,  but  this  slight  amount  is  practically  entirely  con- 
centrated at  the  surface  of  the  water  which  is  in  immediate  contact 
with  the  solid  particles,  owing  to  the  diminution  in  surface  energy 
which  is  thus  brought  about,  so  that  the  litmus  is  only  reddened 
where  it  touches  these  surfaces. 

Uncombined  casein  is  soluble  in  warm  5  per  cent  salt-solution 
and  in  hot  50  per  cent  alcohol.  When  freshly  prepared  and 
warmed  it  is  very  plastic  and  is  capable  of  being  drawn  out  into 
long,  fine  threads  (59). 

Although  it  is  so  insoluble  in  water,  uncombined  casein,  when 
suspended  in  water,  nevertheless  acts  as  an  acid  in  expelling  carbon 
dioxide  from  carbonates  and  bicarbonates,  forming  a  salt  with  the 
base  (51),  and  in  this  way,  according  to  Osborne  (34),  the  ammo- 
nium, potassium,  sodium,  lithium,  magnesium,  strontium  and 
calcium  caseinates  can  be  prepared.  The  salts  of  the  alkaline 


CASEIN  89 

earths  can  be  precipitated  without  alteration  of  their  composition 
by  the  addition  of  alcohol  to  their  solutions  in  water.*  By  direct 
analysis  of  this  precipitate  Van  Slyke  and  Hart  (59)  have  shown 
that  the  calcium  salt  which  is  formed  in  this  way  contains  2.4  per 
cent  of  calcium  oxide  or  from  80  X  10~5  to  90  X  10~5  equivalents  of 
calcium  per  gram  (7).  This  salt,  in  solution,  is  neutral  to  phenol- 
phthalein,  and  the  alcohol  precipitate  from  a  lime-water  solution  of 
casein  which  has  been  neutralized  to  phenolphthalein  contains  ex- 
actly the  same  percentage  of  CaO  (unneutralized  by  mineral  acids) 
as  that  from  the  solution  of  casein  in  a  carbonate-solution.  Similar 
results  had  previously  been  obtained  by  Soldner  (51)  (52),  de  Jager 
(18)  and  Laquer  and  Sackur  (22),  who,  however,  employed  volu- 
metric methods  of  measurement.  Other  6gures  for  the  percentage 
of  CaO  in  this  compound  which  have  been  obtained  by  various 
observers  are:  Courant  (12),  2.91  and  Timpe  (54),  2.62.  These 
figures  are  not  very  divergent,  and  since  the  great  majority  of 
observers  agree  in  placing  the  percentage  at  2.4,  we  may  assume 
that  this  result  is  not  far  from  correct.  The  percentage  of  casein 
in  alkaline  solutions  can  be  fairly  accurately  determined  by  titra- 
tion  to  neutrality  to  phenolphthalein,  upon  the  assumption  that  in 
solutions  of  this  alkalinity  (OH  concentration)  casein  is  combined 
with  2.4  per  cent  of  CaO  or  with  an  equivalent  quantity  of  other 
(strong)  bases. 

If,  however,  a  lime-water  solution  of  casein  be  neutralized  to 
litmus  instead  of  to  phenolphthalein,  by  the  addition  of  mineral 
acid  or  of  casein,  the  alcoholic  precipitate  now  contains  only  1.5 
per  cent  of  CaO  (Van  Slyke  and  Hart  (59)).  The  same  estimate  of 
the  combining-capacity  of  casein  at  absolute  neutrality  (i.e.,  in 
solutions  neutral  to  litmus)  has  been  made  by  Soldner  (51)  (52) 
and  by  myself  (42)  employing  the  potentiometric  method.  The 
casein  from  goats'  milk  combines  with  bases  in  the  same  propor- 
tions as  the  casein  from  cows'  milk  (9).  The  compound  of  casein 
with  2.4  per  cent  of  CaO,  or  an  equivalent  quantity  of  any  other 
strong  base,  is  termed  by  Soldner  and  by  Van  Slyke  and  Hart  the 
''basic"  caseinate,  by  Courant  the  " tricaseinate "  of  the  base. 
That  with  1.5  per  cent  of  CaO,  or  an  equivalent  quantity  of  other 
strong  bases  is  termed  by  Soldner  and  by  Van  Slyke  and  Hart  the 

*  Cf.  Chap.  II.  The  "basic"  salt  of  potassium  (i.e.,  neutral  to  phenol- 
phthalein) is  soluble  in  98.6  per  cent  alcohol,  but  it  may  be  precipitated  from 
this  solution  by  the  addition  of  ether  (45). 


90  CHEMICAL  STATICS 

"neutral"  caseinate;  by  Courant  the  " dicaseinate "  of  the  base. 
Both  terminologies  are  objectionable  since  they  imply  or  suggest 
hypothetical  stoichiometrical  relations.  Neutrality  of  its  solutions 
to  an  arbitrarily  chosen  indicator  is  no  evidence  of  the  unity  of  a 
substance.  As  we  shall  see  in  considering  the  electrochemistry  of 
these  substances,  both  that  containing  2.4  per  cent  CaO  and  that 
containing  1.5  per  cent  CaO  are  mixtures  of  two  or  more  individual 
salts  of  casein.  We  shall,  however,  for  the  sake  of  brevity  in  allu- 
sion, employ  the  nomenclature  of  Soldner,  as  being  the  less  objec- 
tionable of  the  two. 

At  neutrality  either  to  litmus  or  to  phenolphthalein  all  observers 
agree  that  casein  binds  the  alkalies  and  alkaline  earths  in  equiva- 
lent-molecular proportions.  Thus  regular  stoichiometrical  rela- 
tions between  the  protein  and  these  metals  are  clearly  indicated 
although  the  above  cited  data  are  insufficient  to  define  them.  With 
regard  to  ammonium  caseinate,  however,  some  confusion  exists 
since  Bechamp  (3)  states  that  it  contains  1.17  to  1.21  per  cent  of 
ammonia  (=  1.94  to  1.99  per  cent  of  CaO),  while  Salkowski  (47) 
states  that  it  contains  only  0.35  per  cent  of  ammonia.  Salkowski 's 
method  of  preparing  this  compound,  however,  was  faulty  since  he 
precipitated  it  from  alcoholic  solution  by  the  addition  of  NaCl,  a 
procedure  which  involved  the  possibility  of  double  decomposition 
and  the  partial  substitution  of  sodium  for  ammonia  in  the  casein 
compound.* 

The  quantitative  results  for  casein,  which  we  have  so  far  cited, 
may  be  stated  in  other  words  thus:  At  neutrality  to  litmus  one 
gram  of  casein  binds  50  X  10~5  equivalent  gram  molecules  of  a 
base,  and  at  neutrality  to  phenolphthalein  80  X  10~5  equivalent 
gram  molecules. f 

It  was  formerly  stated  by  the  author  (36)  that  if  a  given  con- 
centration of  alkali  be  " saturated"  with  casein,  that  is,  if  it  be 
shaken  up  with  excess  of  casein  until  no  more  of  the  protein  will 
dissolve,  and  then  filtered,  the  filtrate  is  always  neutral  to  litmus 
and  contains  the  quantity  of  casein  required  to  form  the  "neutral" 
caseinate  of  the  base.  In  later  communications  (41)  (42),  I  have 
shown,  however,  that  this  conclusion  was  erroneous,  the  source  of 

*  Regarding  the  actual  occurrence  of  such  types  of  interaction  between 
casein  salts  and  inorganic  salts,  Cf.  W.  A.  Osborne  (35)  and  Van  Slyke  and 
Bosworth  (57). 

t  That  is,  gram  molecules  divided  by  the  valency  of  the  combining  ion. 


CASEIN  91 

error  lying  in  the  fact  that  after  the  attainment  of  neutrality  to 
litmus  additional  casein  dissolves  in  the  solution  with  extreme 
slowness.  Many  hours  of  rapid  shaking  or  stirring  are  quite 
insufficient,  at  room  temperature,  to  bring  about  complete  "  satu- 
ration "  of  the  alkali  with  casein.  Resource  was  had,  therefore,  to 
the  device  of  dissolving  weighed  amounts  of  casein  in  measured 
volumes  of  alkali  of  known  concentration,  and  then  neutralizing 
the  excess  of  alkali  by  the  addition  of  standardized  acid,  stirring 
vigorously  the  while,  until  the  casein  just  began  to  be  precipitated. 
The  point  at  which  precipitation  began  was  readily  determined, 
with  considerable  accuracy,  by  the  change  (diminution)  of  the  re- 
fractive index  of  the  solution.  In  this  way  it  was  ascertained  that 
at  complete  ''saturation"  of  an  alkali  (NaOH  or  LiOH)  one  gram 
of  casein  binds  11.4  X  10~5  equivalent  gram  molecules  of  the  base. 

These  determinations  have  been  repeated  by  Van  Slyke  and 
Bosworth  (57),  whose  results  very  closely  coincide  with  mine. 
They  find  that  at  the  point  at  which  further  diminution  of  the 
proportion  of  base  to  casein  results  in  the  precipitation  of  free 
casein  one  gram  of  casein  is  combined  with  between  11.0  X  10~5 
and  11.5  X  10~5  equivalents  of  ammonium,  sodium  or  potassium. 

These  "saturated"  compounds  of  casein  with  bases  react  in 
solution  to  indicators  as  follows  (41): 

Dimethylaminoazobenzol yellow 

Congo  red red 

Sodium  alizarinsulphonate. red 

Cochineal rose 

Paranitrophenol colorless 

Rosolic  acid yellow 

indicating,  according  to  the  determinations  of  Salm  (48)  an  acidity 
(H+  concentration)  of  about  10~5.  According  to  Michaelis  and 
Rona  (26)  and  Allemann  (1),  the  optimum  reaction  for  the  complete 
precipitation  of  casein  is  2  X  10~5  H+. 

It  is  not  possible  with  equal  simplicity  of  technique  to  determine 
the  combining  capacity  of  casein  for  the  alkaline  earths  at  "satura- 
tion "  of  these  bases  with  casein,  because  the  chloride  of  the  alkaline 
earth,  which  is  necessarily  formed  in  the  solution  at  the  same  time, 
precipitates  the  caseinate.*  Thus  four  grams  of  casein  were  dis- 

*  The  "neutral"  and  "basic"  casemates  of  the  alkaline  earths  are  likewise 
precipitated  by  the  addition  to  their  solutions  of  a  somewhat  greater  propor- 
tion of  the  corresponding  alkaline  earth.  Cf.  A.  S.  Loevenhart  (24)  and  Van 
Slyke  and  Hart  (59). 


92  CHEMICAL  STATICS 

solved  in  100  cc.  of  0.048  N  Ca(OH)2  solution  and  then  40  cc.  of 
AT/10  HC1  were  cautiously  delivered  into  the  solution  by  means  of 
a  pipette  of  which  the  opening  was  held  below  the  surface  of  the 
fluid,  the  fluid  being  rapidly  and  continuously  stirred  meanwhile 
through  the  agency  of  a  small  motor.  The  total  volume  was  then 
made  up  to  200  cc.  and  the  mixture  filtered  through  soft  filter- 
paper.  By  measurement  of  the  refractive  index  of  this  filtrate  it 
was  found  that  the  0.0008  equivalent  of  Ca(OH)2  unneutralized 
by  HC1  had,  under  these  conditions,  only  held  in  solution  0.2 
gram  of  casein;  although,  as  we  have  seen,  in  the  absence  of 
CaCl2,  0.0008  equivalent  of  Ca(OH)2  will  readily  dissolve  one 
gram  of  casein,  rendering  the  solution  neutral  to  phenolphthalein. 
In  a  subsequent  chapter  (X)  it  will  be  shown  that  a  definite 
relationship  subsists  between  the  initial  alkalinity  of  a  solution 
and  the  depression  of  its  electrical  conductivity  which  is  brought 
about  by  the  addition  to  it  of  a  definite  proportion  of  casein.  This 
relationship  is  of  such  a  character  that  by  exterpolation  from  the 
measurements  made  upon  solutions  of  potassium  caseinate  con- 
taining from  25  X  10~5  to  300  X  10~5  equivalents  of  base  per 
gram  of  casein,  it  would  appear  that  the  depression  of  con- 
ductivity caused  by  casein  is  zero  when  the  proportion  of  base  is 
sufficient  and  only  just  sufficient  to  hold  the  casein  in  solution  (42), 
namely,  11.4  X  10~5  equivalents  per  gram.  Similar  exterpola- 
tion from  measurements  of  the  depression  of  the  conductivity  of 
Ca(OH)2  solutions  consequent  upon  the  addition  of  casein  leads  to 
the  conclusion  that  the  depression  of  conductivity  would  vanish 
at  an  initial  alkalinity  corresponding  to  a  proportion  of  11.9  X  10~5 
equivalents  of  Ca(OH)2  per  gram  of  casein,  a  value  so  close  to 
that  obtained  for  the  combining-capacity  of  casein  for  the  alkalies 
at  "saturation"  with  casein  that  I  have  previously  inferred  that 
bases  dissolve  casein  in  equivalent-molecular  proportions  (46). 
Van  Slyke  and  Bosworth  have,  however,  measured  the  combining- 
capacity  of  casein  for  alkaline  earth  bases  at  " saturation"  of  the 
base  with  casein  by  the  more  direct  method  of  first  dissolving  the 
casein  in  an  excess  of  the  base  and  then  neutralizing  the  excess  with 
hydrochloric  acid  and  dialysing  the  mixture  until  free  from  soluble 
chlorides  (57) .  In  this  way-  they  have  found  that  a  compound  of 
casein  with  calcium  hydrate  which  contains  11.25  X  10~5  equiva- 
lents of  base  per  gram  exists  but  is  not  soluble  in  water,  the 
lowest  proportion  of  base  yielding  a  soluble  compound  being 


CASEIN  93 

22.5  X  10~5  equivalents  per  gram,  or  exactly  double  the  pro- 
portion of  alkaline  earth  contained  in  the  insoluble  compound. 
The  insoluble  compound  will,  however,  dissolve  in  solutions  of 
NaCl,  NH4C1,  KC1,  etc.,  probably  owing  to  exchange  of  bases, 
with  the  formation  of  soluble  caseinates  of  the  alkalies  (56)  (57). 

Magnesium  hydrate  forms  a  similar  series  of  compounds  with 
casein,  namely  a  compound  insoluble  in  water  but  soluble  in  5  per 
cent  NaCl  solution  and  containing  11.25  X  10~5  equivalents  of 
base  per  gram,  a  soluble  compound  containing  22.5  X  10~5 
equivalents  of  base  per  gram,  a  compound  neutral  to  litmus 
containing  about  56  X  10~5  equivalents  of  magnesium  per  gram 
and  a  compound  neutral  to  phenolphthalein  containing  between 
80  XlO~5  and  90  X  10~5  equivalents  of  magnesium  per  gram  (62). 

From  the  phosphorus  content  of  casein  (8),  we  would  infer  a 
minimal  combining- weight  of  4430.  The  combining- weight  corre- 
sponding to  the  proportion  of  NaOH  or  KOH  combined  with  casein 
at  "  saturation  "  of  the  base  with  protein  is  8888.  This  corresponds 
so  closely  with  twice  the  minimal  combining-weight  calculated  on 
the  assumption  that  the  casein  molecule  contains  only  one  atom 
of  phosphorus  that  we  may  infer,  with  Bosworth  and  Van  Slyke, 
that  the  molecule  of  casein  contains  two  atoms  of  phosphorus. 

The  maximum  combining-capacity  of  casein  for  bases  (KOH) 
has  been  measured  by  the  author  (42),  employing  the  potentio- 
metric  method.  In  the  presence  of  a  considerable  excess  of  KOH, 
the  combining-capacity  of  casein  attains  the  constant  maximum  of 
180  X  10~5  equivalents  per  gram.  In  passing  from  its  minimum 
to  its  maximum,  therefore,  the  combining-capacity  of  casein  is 
multiplied  sixteen  times. 

The  compounds  of  casein  with  adds  have  not  been  so  extensively 
studied  as  those  with  bases.  Hammarsten  (14)  held  that  there  is 
no  true  combination  between  casein  and  acid,  because  by  pro- 
longed trituration  with  water  he  could  remove  all  of  the  acid 
contained  in  the  casein.  Our  modern  conception  of  balanced 
reactions  deprives  this  consideration  of  its  weight  (59)  (36) .  Since, 
however,  it  has  been  frequently  applied  to  other  protein  com- 
pounds,* it  may  be  worth  while  to  dwell  briefly  upon  the  implica- 
tions of  such  a  deduction. 

*  For  example  by  Hans  Trunkel  (55):  When  so  much  of  the  published 
evidence  on  behalf  of  the  "adsorption  theory"  is  of  this  unfortunate  and 
inconclusive  character,  advocates  of  "adsorption"  cannot  complain  if  their 
hypotheses  are  viewed  with  scepticism  by  physical  chemists. 


94  CHEMICAL  STATICS 

When  any  two  or  more  substances,  for  example,  A  and  B  react 
to  form  other  substances  A'  and  B',  equilibrium  is  reached,  i.e.,  no 
further  change  occurs  in  the  composition  of  the  system,  when  the 
reaction 


proceeds  at  exactly  the  same  velocity  from  left  to  right  as  it 
from  right  to  left.  Since  the  velocity  with  which  a  chemical 
reaction  proceeds  varies  directly  as  the  active  masses  of  the  react- 
ing substances  (Guldberg  and  Waage's  law)  the  velocities  of  the 
two  opposing  reactions  gradually  approach  each  other  as  the 
reaction  proceeds,  until  they  become  equal,  when  the  reaction 
apparently  ceases.  According  to  the  relative  magnitude  of  the 
proportionality-factors  expressing  the  ratio  of  mass  to  velocity  of 
each  of  the  reactions  concerned,  equilibrium  may  occur  so  far  to 
the  left  or  right  that,  with  our  limited  precision  of  measurement, 
the  reaction  appears  to  be  complete  or  not  to  occur  at  all  as  the 
case  may  be,  or  equilibrium  may  occur  when  the  active  masses  of 
the  reacting  components  on  either  side  of  the  equation  are  nearly 
equal;  and,  of  course,  every  type  of  equilibrium  intermediate  be- 
tween these  extremes  is  found  to  occur.  If,  after  the  attainment 
of  equilibrium,  one  of  the  reacting  components  be  wholly  or  par- 
tially removed  from  the  system  the  reaction  will  recommence  more 
or  less  rapidly,  restoring  a  certain  proportion  of  the  abstracted 
component,  and  attaining  a  fresh  station  of  equilibrium  deter- 
mined by  the  new  relative  masses  of  the  components. 

If,  now,  after  the  attainment  of  equilibrium  in  such  a  reaction  as  : 

Casein    +  HC1  <=*  Casein  hydrochloride 

(Insoluble)         (Soluble)  (Insoluble  or  sparingly  soluble) 

even  if  only  a  small  proportion  of  free  HC1  and  casein  exist  in  the 
system,  and  HC1  be  removed  by  trituration  with  water,  the  re- 
action will  recommence,  now  proceeding  from  right  to  left,  and 
restore  a  proportion  of  the  HC1  which  has  been  washed  away.  If 
trituration  be  prolonged  and  the  velocity-constant  (proportionality 
between  mass  and  velocity)  for  the  decomposition  of  the  casein 
hydrochloride  be  tolerably  high,  it  is  easy  to  understand  how  the 
compound  might  ultimately  be  completely  decomposed  and 
deprived  of  its  combined  acid.  Similarly,  the  acid  components  of 
substances  so  universally  admitted  to  be  chemical  compounds  as 
mercuric  sulphate  and  cuprous  chloride  can  be  completely  removed 


CASEIN  95 

from  them  by  prolonged  trituration  with  water  (23)  (16).  To 
argue  from  this  fact  that  these  combinations  are  not  chemical  in 
character  would  be  analogous  to  concluding  that,  because  salicylic 
acid  can  be  completely  removed  from  its  solution  in  water  by 
repeated  extraction  with  ether,  therefore  the  salicylic  acid  was  not 
truly  dissolved  in  the  water. 

To  return  to  the  more  specific  problem  of  the  occurrence  of 
combinations  between  inorganic  acids  and  casein.  Van  Slyke  and 
Hart  (59)  and  Van  Slyke  and  Van  Slyke  (60)  have  described  a 
series  of  compounds  of  casein  with  inorganic  acids  which  are 
insoluble  in  distilled  water.  When  solid  casein  is  suspended  in  a 
dilute  solution  of  an  inorganic  acid,  the  protein  abstracts  some  of 
the  acid  from  the  solution,  forming  these  insoluble  compounds. 
Van  Slyke  and  Van  Slyke  measured  the  velocity  with  which  the 
acid  is  abstracted  from  the  solution  and  the  final  equilibrium  at- 
tained by  abstracting  portions  of  the  well-stirred  suspension,  from 
time  to  time  filtering,  and  measuring  the  electrical  conductivity 
of  the  filtrate.  The  decrease  in  conductivity,  from  the  moment  of 
introduction  of  the  casein,  afforded  a  measure  of  the  amount  of 
acid  bound.  They  found  that  the  acid  is  bound  at  first  rapidly 
and  later  more  slowly  (Cf .  Chap.  XII)  and  at  equilibrium  the  ratio 

acid  bound  by  one  gram  casein 
acid  in  one  cc.  of  solution 

is  nearly  constant  between  the  limits  of  concentration  of  the  acid 
employed  (AT/125  and  Af/lOOO)  for  hydrochloric  acid  at  constant 
temperatures.  When  sulphuric  acid  is  employed  the  ratio  increases 
with  dilution  of  the  acid,  the  quantity  of  acid  bound  by  one  gram 
varying  as  the  square  root*  instead  of  directly  as  the  concentration 
of  the  acid.  The  equilibrium  is  the  same  from  whichever  direction 
it  is  approached,  whether  by  suspending  the  protein  compound  in 
more  dilute  acid  than  that  in  which  it  was  formed,  in  which  case 
it  yields  acid  to  the  solution,  or  by  suspending  uncombined  casein 
directly  in  the  solution  of  acid.  When  one  gram  of  casein  is  sus- 
pended in  100  cc.  of  AT/500  acid  at  0°  C.,  it  takes  up  17.4  X  10~5 
equivalents  of  sulphuric,  11.9  X  10~5  equivalents  of  hydrochloric, 
8.9  X  10~5  equivalents  of  lactic  or  5.3  X  10~5  equivalents  of  acetic 

*  The  exact  exponent  obtained  by  Van  Slyke  and  Van  Slyke  is  1/1.95;  this 
experimental  value  is  so  nearly  |  that  it  affords  no  grounds  for  preferring  the 
experimental  value  to  that  which  is  indicated  by  stoichiometry. 


96  CHEMICAL  STATICS 

acid.  Increase  in  temperature  increases  the  rate  at  which  equi- 
librium is  approached  but  decreases  the  final  amount  of  acid 
taken  up. 

It  is  evident  that  the  concentration  of  acid  was  not  sufficient  in 
any  of  these  solutions  to  bring  about  complete  neutralization  of  the 
casein.  When  more  concentrated  solutions  were  employed,  how- 
ever, a  proportion  of  the  casein  was  dissolved,  and  the  methods  of 
estimation  utilized  by  Van  Slyke  and  Van  Slyke  could  not  be 
employed.  From  considerations  which  follow  below  (Cf.  Chap. 
XII),  it  appears  possible  that  the  compounds  of  casein  with  acids 
which  are  formed  in  experiments  such  as  these  are  in  reality  soluble, 
but  that  while  a  sufficient  proportion  of  casein  remains  uncom- 
bined  they  are  mechanically  hindered  from  passing  out  of  the 
casein  particles  into  the  solvent  (38)  (40).  Van  Slyke  and  Van 
Slyke,  however,  believe  that  the  binding  of  casein  by  acids,  under 
these  conditions,  is  an  "adsorption"  phenomenon  (60)  (61). 

If  one  stirs  excess  of  casein  in  0.1  AT  HC1  (or  more  dilute)  rapidly 
and  continuously  for  over  an  hour,  at  room  temperature  very  little 
or  none  of  the  casein  passes  out  into  the  solvent;  the  solution 
remains  perfectly  clear  on  filtration  and  its  refractive  index  is  only 
very  slightly  changed.  If,  however,  the  casein  be  previously 
dissolved  in  dilute  NaOH  and  then  precipitated  with  HC1,  the 
addition  of  the  slightest  excess  of  HC1  will  then  carry  it  into  solu- 
tion. In  other  words,  wet,  freshly  precipitated  casein  dissolves 
rapidly  in  dilute  acid,  but  the  physical  properties  of  dry,  granular 
casein,  hinder  the  compounds  which  are  formed  from  passing  out 
of  the  particles.*  Dry  serum  globulin  similarly  dissolves  with 
extreme  slowness  in  acids,  although,  when  wet  and  freshly  pre- 
cipitated, it  dissolves  in  dilute  solutions  of  acids  with  great  readi- 
ness. 

I  have  determined  the  acid-equivalent  of  casein  at  " saturation" 
of  the  acid  with  protein  in  the  following  manner  (41):  Weighed 
amounts  of  casein  were  dissolved  in  measured  volumes  of  alkali  of 
known  concentration;  the  excess  of  acid  over  that  sufficient  to 

*  The  fact  that  the  casein  hydrochloride  which  is  formed  is  in  reality  soluble, 
although  it  does  not  pass  out  into  the  solution,  explains  the  dependence  of  the 
amount  formed  upon  the  dilation  of  the  acid,  which  was  observed  by  Van 
Slyke  and  Van  Slyke.  Were  the  casein  salt  insoluble,  as  these  authors  point 
out,  the  amount  formed  should  be  invariable,  or  else  equivalent  to  the  total 
amount  of  acid  in  the  system. 


CASEIN  97 

neutralize  the  alkali,  which  it  was  necessary  to  add  to  the  solution 
in  order  to  just  redissolve  the  casein  was  then  determined.  The 
point  of  complete  re-solution  of  the  casein  was  determined  by 
noting  the  point  at  which  the  refractive  index  of  the  filtered  mix- 
ture attained  a  constant  (maximum)  value.  It  was  found  that  at 
" saturation"  in  solutions  containing  1.25  per  cent  of  casein,  1 
gram  of  casein  =  approximately  32  X  10~5  equivalent  gram  mole- 
cules of  HC1.  The  acid-equivalent  in  more  dilute  solutions  ap- 
pears to  be  somewhat  higher,  although  I  have  not  measured  it 
directly.*  This  is  attributable  to  the  fact  that,  as  the  observa- 
tions of  Van  Slyke  and  Van  Slyke  show,  the  hydrochloride  of 
casein  tends  in  some  measure  to  decompose  into  casein  and  hydro- 
chloric acid,  and,  since  free  casein  is  insoluble,  an  excess  of  acid 
over  that  actually  combined  with  the  casein  is  necessary  in  order 
to  diminish  this  (hydrolytic)  dissociation  sufficiently  to  keep  the 
casein  in  solution.  The  same  phenomenon,  as  we  shall  see,  is 
encountered  in  acid  solutions  of  serum  globulin. 

Schlossmann  (50)  and  Arny  and  Pratt  (2)  have  described  com- 
pounds of  casein  with  alums  which,  especially  that  with  ferric 
alum,  are  but  sparingly  soluble.  Upon  this  fact  Arny  and  Pratt 
have  based  a  method  of  estimating  casein  volumetrically. 

Paracasein,  the  product  of  the  action  of  rennet  upon  casein, 
resembles  casein  very  closely,  save  in  the  greater  insolubility  of 
the  calcium  salt  in  the  presence  of  an  excess  of  calcium  ions. 
Calcium  caseinate  is  precipitated  by  calcium  chloride,  a  smaller 
amount  of  calcium  chloride  being  required  to  bring  about  complete 
precipitation,  the  smaller  the  proportion  of  base  in  the  caseinate 
(i.e.,  the  more  acid  the  solution).  This  precipitation  is  still  more 
readily  accomplished  at  higher  temperatures  (24)  (59).  Calcium 
paracaseinate  differs  from  calcium  caseinate  in  that  it  is  precipi- 
tated by  a  smaller  excess  of  calcium  ions,  and,  by  a  given  excess, 
at  a  lower  temperature  (24).  Paracasein  differs,  however,  still 
more  markedly  from  casein  in  the  proportion  of  base  which  it 
binds  at  " saturation"  of  the  base  with  protein,  i.e.,  when  the 
proportion  of  inorganic  base  is  sufficient  and  only  just  sufficient  to 

*  The  accuracy  of  this  estimate  of  the  combining-capacity  of  casein  for 
acids  at  "saturation"  of  the  acid  by  protein  cannot  be  relied  upon,  since  the 
solution  necessarily  contained  a  certain  quantity  of  the  chloride  of  the  alkali 
employed  to  dissolve  the  casein,  and  the  compounds  of  casein  with  acids  are 
very  readily  precipitated  by  salts. 


98 


CHEMICAL  STATICS 


hold  the  paracasein  in  solution.  We  have  seen  that  the  proportion 
of  alkali  which  just  suffices  to  hold  casein  in  solution  in  water  is 
about  11.25  X  10~5  equivalents  per  gram.  Paracasein,  however, 
requires  exactly  double  this  proportion  of  alkali  to  hold  it  in 
solution  (57)  indicating,  as  Van  Slyke  and  Bosworth  believe,  that 
the  molecule  of  paracasein  is  one-half  the  weight  of  the  molecule 
of  casein.  From  this  it  would  follow  that  the  molecule  of  para- 
casein contains  but  one  atom  of  phosphorus  (58). 

4.  The  Compounds  of  Serum  Globulin  with  Inorganic  Acids 
and  Bases.  —  The  compounds  of  the  globulin  which  is  precipi- 
tated from  diluted  serum  by  the  passage  of  C02  through  it,  and 
which,  in  the  free  condition,  is  insoluble  in  distilled  water,  have 
been  extensively  studied  by  W.  B.  Hardy  (15).  This  investigator 
has  measured  the  quantities  of  various  acids  which  are  required 
to  dissolve  one  gram  of  globulin  at  various  concentrations.  The 
mean  values,  measured  in  gram  molecules,  taking  the  quantity  of 
HC1  required  to  dissolve  one  gram  of  the  globulin  as  unity,  are 
quoted  below: 

HC1 1.0 

HNO3 0.995 

CHC12COOH 1.0 

CH2C1COOH....1.05 

HCOOH 1.25 

CHaCOOH 5.2 

CH3CH2COOH...7.56 

It  is  evident  that  the  acid-equivalent  of  serum-globulin  is  the 
same  for  all  of  the  strong  monobasic  acids  investigated,  although 
it  is  much  higher  for  the  weak  acids.  This  equivalent  is,  for  one 
gram  of  the  protein,  about  18  X  10~5  gram-equivalents  (mean 
value).  It  is,  however,  somewhat  higher  the  more  dilute  the 
globulin,  and  this  effect  is  more  marked  with  the  weak  than  with 
the  stronger  acids.  Thus : 


H2SO4 1.91       Citric... 

Tartaric 1 . 994     H3PO4 .  . 

Oxalic..  ..1.9        H3BO3.. 


3 

2.9 

,    .  very  great 
excess 


Concentration  of  protein, 
per  cent 

Gram-equivalents  of  acid  necessary  to  dissolve  one 
gram  of  protein 

Acetic  acid 

Hydrochloric  acid 

0.28 
1.46 
4.18 

183  X10-5 
56X10~5 
40X10-6 

23X10-* 
15X10-5 
13X10-6 

SERUM   GLOBULIN 


99 


We  have  already  had  occasion  to  discuss  the  interpretation  of  simi- 
lar phenomena  which  are  displayed  by  the  compounds  of  casein 
with  acids. 

The  acid-equivalent  of  serum-globulin  for  dibasic  (strong)  acids 
is  twice  that  for  monobasic  acids,  and  for  tribasic  (strong)  acids 
it  is  three  times  that  for  monobasic  acids.  Hence  serum-globulin 
combines  with  acids,  at  "saturation"  in  molecular  and  not  equiva- 
lent molecular  proportions. 

Solutions  of  acids  " saturated"  with  serum-globulin  are  faintly 
acid  to  methyl  orange.  • 

Employing  the  potentiometric  method  (37)  (39)  I  have  deter- 
mined the  quantity  of  acid  neutralized  by  globulin  in  solutions  of 
hydrochloric  acid  of  various  concentrations.  The  following  are 
among  my  results,  the  concentration  of  globulin  in  each  solution 
being  0.496  per  cent. 


Concentrations  of  HC1  in 
which  globulin  was  dissolved 

Concentration  of  H     ions  in 
solution  containing  globulin 

Equivalents  of  HC1  neutral- 
ized per  gram  of  globulin 

38.5  X  10-4 
57.8  X  10~4 
77.0  X  10~4 
120.3  X  10~4 

12.4  X  10~4 
23.8  X  10~4 
36.8  X  10~4 
72.7  X  10~4 

53  X  10~5 
69  X  10~5 
81  X  10~5 
96  X  10~5 

We  see  that  the  acid-equivalent  of  serum-globulin  rises  with  in- 
creasing acidity  of  its  solution. 

The  compounds  of  serum-globulin  with  bases  have  also  been 
investigated  by  Hardy.  He  finds  that  at  "  saturation  "  of  solutions 
of  bases  with  serum-globulin  the  bases  are  bound  by  the  globulin 
in  molecular,  not  equivalent  molecular  proportions.  Solutions  of 
monacid  bases  " saturated"  with  globulin  are  neutral  to  litmus, 
the  alkali  equivalent  for  monacid  bases  being  approximately 
10  X  10~5  gram-equivalents  of  the  base  per  gram  of  globulin,  that 
for  di-acid  bases  being  approximately  20  X  10~5  in  the  same  units. 

At  neutrality  to  phenolphthalein,  however,  that  is,  at  an  alka- 
linity corresponding  to  an  OH'  concentration  of  20  X  10~7  (Cf. 
Salm  (48)),  the  bases  are  bound  in  equivalent  molecular  pro- 
portions, such  that  one  gram  of  globulin  binds  20  X  10~5  gram- 
equivalents  of  the  base.  Thus  the  point  of  complete  saturation  of 
Ba(OH)2  solutions  by  globulin  and  that  of  neutrality  to  phenol- 
phthalein coincide,  while  solutions  of  the  monacid  bases  which  are 


100  CHEMICAL  STATICS 

saturated  with  globulin  contain  only  half  the  proportion  of  base 
to  protein  which  is  contained  in  those  which  are  neutral  to  phenol- 
phthalein.  Hardy  has  suggested,  in  explanation  of  this,  that 
globulin  has  at  least  two  replaceable  hydrogens  and  that  its  acid 
salts  of  sodium  or  potassium  are  soluble,  while  its  acid  salt  of 
barium  is  relatively  insoluble,  the  "  neutral "  salt  being  soluble 
and  neutral  to  phenolphthalein.  Since  complete  solution  of  the 
globulin  is  just  attained  at  neutrality  to  litmus  and  the  ratio 
of  the  alkali-binding  capacity  of  serum  at  neutrality  to  phenol- 
phthalein to  its  capacity  at  neutrality  to  litmus  is  2:1,  this 
hypothesis,  although  based  only  upon  results  obtained  with  arbi- 
trarily chosen  indicators,  would  appear  to  be  justifiable.  We 
shall  have  occasion  to  further  discuss  this  question  from  a  some- 
what different  point  of  view  in  Chap.  IX. 

Employing  the  potentiometric  method  (37)  I  have  determined 
the  quantity  of  alkali  neutralized  by  globulin  in  solutions  contain- 
ing an  excess  of  KOH.  The  following  were  the  results  obtained, 
the  concentration  of  globulin  in  each  solution  being  0.208  per  cent. 


Concentration  of  KOH  in  which 
globulin  was  dissolved 

Equivalents  of  KOH  bound  by 
one  gram  of  globulin 

12.9  X  10~4 
16.1  X  10~4 
19.3  X  lO-4 
25.8  X  10~4 

47  X  10-5 
46  X  10~5 
50  X  10~5 
45  X  10-* 

The  alkali-equivalent  of  the  globulin,  therefore,  remained  appreci- 
ably constant  throughout  the  range  of  alkalinities  mentioned.  Its 
mean  value  is  47  X  10~5  gram-equivalents  of  KOH  per  gram 
of  globulin,  which  may  therefore  be  regarded  as  the  alkali-equiva- 
lent of  globulin  in  the  presence  of  excess  of  base. 

The  ratio  of  the  alkali  to  the  acid-equivalent  of  globulin  at 
"saturation"  is,  according  to  Hardy's  determinations  10  :  18.  He 
considers  that  the  true  ratio  is  probably  1  :  2. 

The  compounds  of  globulin  with  salts  have  been  studied  by 
Hardy  (15)  and  by  Mellanby  (25).  But  as  these  compounds  have 
not  yet  been  clearly  defined,  and  their  discussion  involves  a  con- 
sideration of  the  general  nature  of  the  mechanism  of  the  solution 
and  precipitation  of  protein  by  salts,  the  description  of  the  results 
obtained  by  these  observers  will  be  deferred  to  the  succeeding 
chapter. 


VEGETABLE  PROTEINS  •'  '> .  '<  : ^  10 1  / 

6.  The  Compounds  of  Fibrin  with  Inorganic  Acids  and  Bases. 
—  Employing  methods  similar  to  those  utilized  by  Van  Slyke  and 
Bosworth  in  the  investigation  of  the  compounds  of  casein  with  in- 
organic bases,  Bosworth  has  investigated  the  combining-capacity 
of  fibrin  for  inorganic  bases  and  acids  (6).  He  finds  that  at  neu- 
trality to  phenolphthalein  one  gram  of  fibrin  neutralizes  61.4  X 
10~5  equivalents  of  NaOH  or  61.6  X  10~5  equivalents  of  Ca(OH)2. 
Fibrin,  therefore,  neutralizes  bases,  in  the  presence  of  excess  of  the 
base,  in  equivalent-molecular  proportions.  At  "  saturation,"  i.e., 
when  the  proportion  of  base  is  only  just  sufficient  to  hold  the  pro- 
tein in  solution,  fibrin  combines  with  15.2  X  10~5  equivalents  of 
NaOH,  and  the  precipitate  produced  on  addition  of  HC1  to  this 
solution  is  free  from  sodium.  In  other  words  no  insoluble  salts  of 
sodium  are  formed.  The  case  is  quite  otherwise  with  calcium 
which  forms  with  fibrin  an  insoluble  salt  (soluble,  however,  in 
5  per  cent  NaCl  solution),  containing  30  X  10~5  equivalents  of  the 
base,  and  a  soluble  salt  containing  45  X  10~5  equivalents  of  the 
base.  The  proportion  of  1:2:3  encountered  in  these  various 
compounds  is  irresistible  evidence  of  the  existence  of  definite 
stoichiometrical  relations  between  fibrin  and  the  bases  with  which 
it  combines. 

The  sulphur-content  of  fibrin  would  indicate  a  minimal  com- 
bining-weight  of  6751.  The  proportion  of  base  in  the  " saturated" 
compound  of  fibrin  with  NaOH  would  indicate  a  combining-weight 
of  6667. 

Calcium  and  barium  fibrinates  yield  a  precipitate  of  fibrin  on  the 
passage  of  CO2  through  them  (6)  (17),  sodium,  potassium  and 
ammonium  fibrinates  do  not.  Fibrin  does  not  decompose  CaCO3, 
however,  as  casein  does  on  trituration  with  this  salt  in  the  presence 
of  water. 

Fibrin  forms  a  compound  with  hydrochloric  acid,  which  is  just 
soluble  in  water,  containing  15  X  10~5  equivalents  of  the  acid. 

6.  The  Compounds  of  the  Vegetable  Proteins  with  Inorganic 
Acids  and  Bases.  —  These  have  been  investigated  by  T.  B. 
Osborne  (29)  (30)  (31)  (32),  who  has  found  evidence  of  combina- 
tion between  inorganic  acids  and  the  following  vegetable  proteins : 
Edestin,  legumin,  excelsin,  amandin,  corylin,  phaseolin,  gliadin, 
hordein  and  zein.  He  finds  that  when  edestin,  deposited  from 
salt-solutions,  is  suspended  in  water  and  made  neutral  to  phenol- 
phthalein, edestin  itself  remains  undissolved,  while  the  added 
alkali  carries  into  solution  its  equivalent  of  the  acid  or  acids  which 


102  CHEMICAL  STATICS 

had  been  united  with  the  edestin.  The  character  of  'the  acid 
united  with  the  edestin  varies  with  that  of  the  salt  employed  for 
solution  of  the  edestin.  When  the  salt  is  sodium  chloride,  the  acid 
is  HC1,  when  it  is  ammonium  sulphate,  the  acid  is  H2S04.  The 
combination  is  apparently  facilitated  by  a  weakly  acid  reaction, 
not  necessarily  induced  by  the  same  acid  as  that  with  which  the 
edestin  combines.  Osborne  attributes  this  phenomenon  to  the 
strength  of  edestin  as  a  base;  the  compound  which  is  formed, 
however,  must  be  practically  undissociated  so  far  as  Cl'  ions  are 
concerned,  otherwise,  having  regard  to  the  extremely  low  equiv- 
alent-concentration of  protein  in  the  solutions  in  which  it  is 
formed,  we  should  attribute  to  edestin  a  basic  function  of  a  magni- 
tude wholly  inconsistent  with  the  fact  that  it  is  an  amphoteric 
acid.  Therefore,  it  does  not  follow  that  these  proteins  are  stronger 
bases  than  they  are  acids  because  they  can  displace  hydrochloric 
acid  from  its  combination  with  sodium  hydrate,  as  Osborne 
assumes,  for  this  phenomenon  may  be  interpreted  by  merely 
supposing  that  the  protein  can  combine  with  either  constituent 
of  the  organic  salt,  but  that  the  compound  with  HC1  produces 
fewer  Cl'  ions  than  the  Na+  ions  produced  by  the  compound  with 
NaOH.  A  further  discussion  of  this  phenomenon  will  be  found  in 
a  later  chapter.  (Chap.  VI,  section  6.) 

The  fact  that  the  occurrence  of  decomposition  of  neutral  mineral 
salts  through  the  agency  of  animal  proteins  has  not  been  generally 
recognized,  is  probably  attributable  to  the  fact  that  it  has  not  been 
carefully  looked  for,  and  that  the  proteins  which  are  most  readily 
obtained  in  the  pure  condition  are  not  usually  prepared  under 
conditions  favoring  the  formation  of  protein  compounds  through 
the  decomposition  of  inorganic  salts. 

Two  compounds  of  edestin  with  acid  (hydrochloric)  appear  to 
exist.  The  one,  which  Osborne  terms  the  monohydrochloride, 
contains  an  amount  of  acid-equivalent  to  0.7  cc.  N/IQ  HC1  per 
gram  of  the  protein,  and  is  insoluble  in  water;  it  is,  however, 
soluble  in  salt  solution  and  is  deposited  therefrom  in  crystals  on 
dialysis.  The  other  compound  of  edestin  with  hydrochloric  acid 
contains  just  sufficient  acid  to  hold  the  protein  in  solution,  and  is 
termed  by  Osborne  the  dihydrochloride.  The  quantity  of  HC1 
required  to  just  hold  one  gram  of  edestin  in  solution  is  1.4  cc. 
of  AT/10  HC1.  The  acid-equivalent,  for  monobasic  acids,  at 
"  saturation "  of  the  acid  with  edestin  is  therefore  14  X  10~5 
gram-equivalents  per  gram.  The  quantity  of  alkali  which  will 


SUMMARY  103 

just  hold  one  gram  of  edestin  in  solution  is  equivalent  to  the 
quantity  of  acid  with  which  it  combines  to  form  Osborne' s  "edes- 
tin monohydrochloride,"  that  is,  7  X  10~5  equivalents.  Hence  the 
ratio  of  the  alkali-  to  the  acid-equivalent  at  "  saturation  "  is  1  :  2. 

From  the  observations  of  Osborne  it  would  appear  that  acids 
dissolve  edestin,  as  they  do  serum-globulin,  in  molecular  propor- 
tions, not  in  equivalent  molecular  proportions. 

Osborne  has  also  determined  the  hydrochloric  acid  equivalents 
at  neutrality  to  tropceolin  (10~2  to  10~3  H+  according  to  Salm 
(48)),  of  a  variety  of  vegetable  proteins.  That  of  edestin  was 
found  to  be  127  X  10~5  gram-equivalents  per  gram,  of  excelsin 
124  X  10-5,  of  legumin  129  X  10~5,  of  amandin  103  X  10~5,  of 
crystallized  egg-albumin  90  X  10~5  in  the  same  units.  Recol- 
lecting that  these  determinations  are  made  in  the  presence  of  a 
considerable  excess  of  acid  they  may  be  considered  as  furnishing, 
possibly,  a  measure  of  the  constant  maximum  acid-equivalents  of 
these  proteins  in  the  presence  of  excess  of  acid.  However  this  may 
be,  it  is  evident  that  the  acid-equivalent  of  edestin  increases  very 
considerably  with  the  acidity  of  its  solution. 

The  compounds  which  edestin  and  gliadin  form  with  cupric 
hydroxide  (33)  have  already  been  discussed  in  Chap.  I. 

7.  The  Compounds  of  Ovomucoid  with  Acids  and  Bases.  — 
These  have  so  far  chiefly  been  studied  by  electrochemical  methods 
(43).     Uncombined  ovomucoid  is  freely  soluble  in  distilled  water, 
but  on  adding  small  quantities  of  acid  or  alkali  to  this  solution 
a  proportion  of  the  acid  or  base  is  neutralized  by  the  protein. 
Employing  the  potentiometric  method  I  have  shown  that  at  neu- 
trality to  litmus  (i.e.,  absolute  neutrality)  1  gram  of  ovomucoid 
neutralizes  7  X  10~5  equivalents  of  HC1.    Its  basic  function,  there- 
fore, predominates  over  its  acid  function. 

The  maximum  (constant)  combining-capacity  of  ovomucoid  for 
KOH,  in  solutions  containing  excess  of  the  base,  is  about  50  X  10~5 
equivalents  per  gram. 

The  maximum  combining-capacity  of  ovomucoid  for  HC1  was 
not  attained  in  any  of  the  solutions  investigated,  but  is  probably 
in  excess  of  100  X  10~5  equivalents  per  gram. 

In  the  chapters  dealing  with  the  electrochemistry  of  the  proteins 
the  reader  will  find  a  more  detailed  discussion  of  these  compounds. 

8.  Summary  of  Some  of  the  Results  Cited  in  this  Chapter.  — 
Some  of  the  more  important  determinations  which  have  been  cited 
in  this  chapter  are  summarized  in  the  following  table: 


104 


CHEMICAL  STATICS 


ill 

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«a^ 

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E'o 


>     -° 

ISs 


la    a 

£.9        SS 


>0  rH 


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2S 
X"o 


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S.        -i 

u.  I 


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I 
f5     8 

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LITERATURE  CITED  105 


LITERATURE  CITED 

(1)  Allemann,  O.,  Biochem.  Zeit.  45  (1912),  p.  346. 

(2)  Arny,  H.  V.,  and  Pratt,  T.  M.,  Amer.  Journ.  of  Pharmacy,  78  (1906), 

p.  121. 

(3)  Be'champ,  A.,  Bull.  Soc.  Chem.  (3)  11  (1894),  p.  153. 

(4)  Bosworth,  A.  W.,  Journ.  Biol.  Chem.  15  (1913),  p.  231. 

(5)  Bosworth,  A.  W.,  Journ.  Biol.  Chem.  19  (1914),  p.  397. 

(6)  Bosworth,  A.  W.,  Journ.  Biol.  Chem.  20  (1915),  p.  91. 

(7)  Bosworth,  A.  W.,  and  Van  Slyke,  L.  L.,  Journ.  Biol.  Chem.  14  (1913), 

p.  207. 

(8)  Bosworth,  A.  W.,  and  Van  Slyke,  L.  L.,  Journ.  Biol.  Chem.  19  (1914), 

p.  67. 

(9)  Bosworth,  A.  W.,  and  Van  Slyke,  L.  L.,  Journ.  Biol.  Chem.  24  (1916), 

p.  173. 

(10)  Burian,  R.,  Ergeb.  d.  physiol.  5  (1906),  p.  768. 

(11)  Cohnheim,  O.,  "Chemie  der  Eweisskorper  "  (1900),  p.  173. 

(12)  Courant,  G.,  Arch.  f.  d.  Ges.  physiol.  50  (1891),  p.  109. 

(13)  Goto,  M.,  Zeit.  f.  physiol.  Chem.  37  (1902),  p.  94. 

(14)  Hammarsten,  O.  Nov.  Act.  Reg.  Soc.  Upsala  (1877),  cited  after  Maly's 

Jahresber.  f.  Tierchem.  (1877),  p.  158. 

(15)  Hardy,  W.  B.,  Journ.  of  Physiol.  33  (1905),  p.  251. 

(16)  Haywood,  J.  K.,  Journ.  of  Physical  Chem.  1  (1897),  p.  411. 

(17)  Hekma,  E.,  Koninklijke  Akad.  Van  Wetenschappen  Te  Amsterdam 

16  (1913),  p.  172. 

(18)  De  Jager,  L.,  Nederl.  Tijdschr.  Voor  Geneeskunde.  2  (1897),  p.  253, 

cited  after  Maly's  Jahresber.  f.  Tierchem.  27  (1897),  p.  276. 

(19)  Kossel,  A.,  Zeit.  f.  physiol.  Chem.  25  (1898),  p.  165. 

(20)  Kossel,  A.,  and  Weiss,  F.,  Zeit.  f.  physiol.  Chem.  78  (1912),  p.  402. 

(21)  Kurajeff,  D.,  Zeit.  f.  physiol.  Chem.  26  (1899),  p.  524. 

(22)  Laqueur,  E.,  and  Sackur,  O.,  Beitr.  z.  chem.  Physiol.  und  Pathol.  3 

(1902),  p.  193. 

(23)  Lescoeur,  H.,  Ann.  de  chim.  et  de  phys.  7  Ser.  2  (1884),  p.  78. 

(24)  Loevenhart,  A.  S.,  Zeit.  f.  physiol.  Chem.  41  (1904),  p.  177. 

(25)  Mellanby,  J.,  Journ.  of  Physiol.  33  (1905),  p.  338. 

(26)  Michaelis,  L.,  and  Rona,  P.,  Biochem.  Zeit.  28  (1910),  p.  197. 

(27)  Miescher,  F.,  Verhand.  Naturforsch.  Ges.  in  Basle  6  (1874),  p.  138. 

(28)  Miescher,  F.,  Arch.  f.  Exper.  Path,  und  Pharm.  37  (1896),  p.  100. 

(29)  Osborne,  T.  B.,  Journ.  Amer.  Chem.  Soc.  21  (1899),  p.  486. 

(30)  Osborne,  T.  B.,  Zeit.  f.  physiol.  Chem.  33  (1901),  p.  240. 

(31)  Osborne,  T.  B.,  "The  Vegetable  Proteins,"  London  (1909). 

(32)  Osborne,  T.  B.,  Ergeb.  d.  physiol.  10  (1910),  p.  47. 

(33)  Osborne,  T.  B.,  and  Leavenworth,  C.  S.,  Journ.  Biol.  Chem.  28  (1916), 

p.  109. 

(34)  Osborne,  W.  A.,  Journ.  of  Physiol.  27  (1901),  p.  398. 

(35)  Osborne,  W.  A.,  Proc.  physiol.  Soc.;  Journ.  of  Physiol.  33  (1905),  p. 

10;  34  (1906),  p.  84. 

(36)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  2  (1907),  p.  317. 


106  CHEMICAL  STATICS 

(37)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  11  (1907),  p.  437. 

(38)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  4  (1908),  p.  35. 

(39)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  5  (1908),  p.  155. 

(40)  Robertson,  T.  Brailsford,  Zeit.  f.  Kolloidchem.  3  (1908),  Heft.  2. 

(41)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  13  (1909),  p.  469. 

(42)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  14  (1910),  p.  528. 

(43)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  14  (1910),  p.  709. 

(44)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  15  (1911),  p.  179. 

(45)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.  15  (1911),  p.  387. 

(46)  Robertson,  T.  Brailsford,  "Die  physikalische  Chemie  der  Proteine," 

Dresden  (1912),  p.  71. 

(47)  Salkowski,  E.,  Zeit.  f.  Biol.  37  (1899),  p.  404. 

(48)  Salm,  E.,  Zeit.  f.  physikal.  Chem.  57  (1906),  p.  471. 

(49)  Samuely,  F.,  Oppenheimer's  Handbuch  der  Biochem.  (1909),  Bd.  1, 

p.  35. 

(50)  Schlossmann,  A.,  Zeit.  f.  physiol.  Chem.  22  (1896),  p.  197. 

(51)  Soldner,  F.,  Landwirthschaft.  Versuchst.  35  (1888),  p.  351. 

(52)  Soldner,  F.,  Zeit.  f.  angew.  Chem.  (1895),  p.  370. 

(53)  Taylor,  A.  E.,  Univ.  of  California  Publ.  Pathol.  1  (1904),  p.  7. 

(54)  Timpe,  H.,  Arch.  f.  Hyg.  18  (1893),  p.  1. 

(55)  Trunkel,  H.,  Biochem.  Zeit.  26  (1910),  p.  493. 

(56)  Van  Slyke,  L.  L.,  and  Bosworth,  A.  W.,  New  York  Agric.  Expt.  Stn. 

Technic.  Bull.  Nr.  26  (1912). 

(57)  Van  Slyke,  L.  L.,  and  Bosworth,  A.  W.,  Journ.  Biol.  Chem.  14  (1913), 

p.  211. 

(58)  Van  Slyke,  L.  L.,  and  Bosworth,  A.  W.,  Journ.  Biol.  Chem.  14  (1913), 

p.  227. 

(59)  Van  Slyke,  L.  L.,  and  Hart,  E.  B.,  Amer.  Chem.  Journ.  33  (1905),  p.  461. 

(60)  Van  Slyke,  L.  L.,  and  Van  Slyke,  D.  D.,  Amer.  Chem.  Journ.  38  (1907), 

p.  383. 

(61)  Van  Slyke,  L.  L.,  and  Van  Slyke,  D.  D.,  Journ.  Biol.  Chem.  4  (1908), 

p.  259. 

(62)  Van  Slyke,  L.  L.,  and  Winter,  O.  B.,  Journ.  Biol.  Chem.  17  (1914),  p. 

287. 


CHAPTER  VI 
THE   COMPOUNDS   OF  THE  PROTEINS    (Continued) 

1.  General  Remarks  on  the  Precipitation  of  Proteins  by 
Inorganic  Salts.  —  It  was  pointed  out  by  Hardy  (24)  in  his 
exhaustive  communication  on  globulin  that  the  precipitation 
of  proteins  and,  indeed,  of  colloids  in  general  may  be  of  two 
kinds.  The  first  is  clearly  accompanied  by  decomposition  of 
the  precipitating  agent;  it  will  not  occur,  as  Pauli  has  demon- 
strated (56)  unless  the  protein  is  in  some  proportion  ionized, 
and  relatively  small  quantities  of  the  precipitating  agent  are 
required  to  bring  about  the  precipitation.  The  second  kind  of 
precipitation,  however,  whether  accompanied  by  decomposition 
of  the  precipitating  agent  or  not,  occurs  even  when  the  protein 
is  non-ionic  and  requires  relatively  large  amounts  of  the  pre- 
cipitating agent.  Precipitation  of  the  first  kind  is,  generally 
speaking,  only  brought  about  by  electrolytes,  while  precipitation 
of  the  second  kind,  although  as  a  rule,  more  readily  brought 
about  by  electrolytes  than  by  non-electrolytes,  may  nevertheless 
be  brought  about  by  certain  non-electrolytes,  for  example,  by 
alcohol. 

For  this  latter  type  of  precipitation  we  shall  henceforth,  when- 
ever possible,  reserve  the  term  coagulation.*  Both  precipitation 
and  coagulation  of  a  protein  may  be  brought  about  by  one  and 
the  same  inorganic  salt.  In  such  a  case  the  gradual  addition  of 
salt  to  the  originally  salt-free  solution  which  contains  ionic  pro- 
tein, i.e.,  protein  which  drifts  to  one  electrode  or  to  the  other  in 
an  electric  field,  first  brings  about  precipitation  and  then  reso- 
lution of  the  protein.  In  this  new  solution  the  protein  is,  accord- 

*  Much  confusion  exists  in  the  literature  on  this  subject  on  account  of  the 
fact  that  the  distinction  between  the  precipitation  of  a  protein  through  chemical 
interaction  with  the  added  salt,  and  its  coagulation  through  the  change  in  the 
nature  of  the  solvent  resulting  from  the  further  addition  of  salt  has  not  invari- 
ably been  recognized. 

107 


108  CHEMICAL  STATICS 

ing  to  Hardy,  non-ionic  and  it  can  be  coagulated  by  still  further 
addition  of  the  salt.* 

The  first  kind  of  precipitation  appears  to  be  undoubtedly 
chemical  in  character  and  in  mechanism.  The  mechanism  of 
coagulation  is,  however,  far  from  clear,  and  for  the  attainment 
of  an  adequate  understanding  of  this  phenomenon  we  shall 
doubtless  have  to  wait  until  the  physico-chemical  theory  of  solu- 
tions in  general  has  reached  a  more  mature  stage  of  development 
than  it  has  at  present.  At  least  three  possibilities  exist. 

(i)  The  coagulation  of  proteins  by  salts  is  a  purely  physical 
phenomenon  due  either  to  an  alteration  in  the  electrical  condition 
of  the  protein  (Bredig,  Billitzer,  Freundlich)  or  to  a  physical 
alteration  in  the  nature  of  the  solvent. 

(ii)  The  coagulation  of  proteins  by  salts  is  partly  a  physical 
and  partly  a  chemical  phenomenon  depending  upon  the  forma- 
tion of  various  compounds  between  the  protein  and  the  salt, 
and  upon  their  varying  solubilities  in  salt  solutions  (Spiro, 
Galeotti). 

(iii)  The  coagulation  of  proteins  by  salts  is  indirectly  a  chemi- 
cal phenomenon,  attributable  to  a  disturbance  in  the  chemical 
equilibrium  between  the  protein  and  its  solvent  (Hofmeister, 
Pauli). 

In  reviewing  the  various  factors  which  have  been  ascertained 
to  be  of  importance  in  determining  the  precipitation  and  coagu- 
lation of  proteins  by  electrolytes  we  shall  incidentally  discuss  the 
applicability  of  these  several  hypotheses. 

2.  Earlier  Investigations  on  the  Significance  of  the  State  of 
Hydration  of  the  Proteins  in  Relation  to  their  Coagulation  by 
Salts.  —  The  fact  that  proteins  can  be  thrown  out  of  solution  by 
the  addition  thereto  of  inorganic  salts  appears  to  have  first  been 
pointed  out  by  Claude  Bernard  (2),  who  employed,  among  others, 
magnesium  sulphate,  sodium  sulphate  and  ammonium  carbonate. 
As  early  as  1854,  Virchow  (76)  suggested  that  the  inorganic 

*  An  interesting  example  of  the  dependence  of  the  ionization  of  a  protein 
upon  concentration  (i.e.,  available  mass  of  water)  of  the  medium  in  which  it 
is  dissolved  is  afforded  by  serum-globulin  dissolved  in  solutions  of  sodium 
citrate.  In  solutions  containing  low  concentrations  of  sodium  citrate  the 
protein  is  ionized  and  drifts  in  an  electric  field,  while  the  same  protein  when 
dissolved  in  more  concentrated  sodium  citrate  solution  is  found  to  be  no  longer 
ionized  (-9). 


EARLIER  INVESTIGATIONS 


109 


salts  render  proteins  insoluble  by  extracting  water  from  them. 
This  suggestion  was  again  put  forward  in  1888  by  F.  Hofmeister 
(30),  who,  together  with  his  pupils,  advanced  in  the  immediately 
succeeding  years,  a  number  of  experimental  data  in  support  of 
his  thesis  (30)  (31)  (38)  (62)  (43). 

Kiihne  (39)  and  Kauder  (38),  having  shown  that  not  only  the 
concentration  of  the  added  salt  but  also  that  of  the  protein  is 
of  importance  in  determining  the  amount  of  salt  required  for  its 
coagulation,  care  was  taken  in  these  and  in  the  majority  of  suc- 
ceeding investigations,  when  comparing  the  coagulating  power  of 
different  salts,  to  maintain  the  concentration  of  the  protein 
constant,  the  procedure  being  to  add  to  a  given  volume  of  protein- 
containing  fluid  (e.g.,  blood  serum)  varying  amounts  of  different 
salt  solutions,  and  then  dilute  the  mixture  to  a  standard  volume. 

Lewith  showed  that  the  relative  efficacy  of  the  different  salts 
which  he  employed  in  coagulating  the  proteins  of  blood  serum 
was  the  same  for  serum  globulins  as  for  serum-albumins,  sul- 
phates and  acetates  being  more  powerful  coagulants  than  nitrates 
or  chlorides.  His  results  have  been  tabulated  as  follows  by 
Gustav  Mann  (46),  a  (+)  indicating  that  the  salts  do,  and  a  (  — ) 
that  they  do  not  precipitate  serum-albumin. 


Potassium 

Sodium 

Ammonium 

Magnesium 

Calcium 

Barium 

Acetate  

+ 

+ 

Chloride.     .    .. 

+ 

+ 





+ 

, 

Nitrate  

+ 





i 

^^ 

Phosphate 

+ 

Sulphate 

_ 

+ 

-f 

+ 

Sulphocyanate 

Iodide  













Bromide 













Chromate 

_ 

Bicarbonate 

_ 

Hofmeister,  in  1888,  extended  and  confirmed  these  observations, 
employing  not  only  serum-proteins  but  also  egg-albumin,  gelatin 
and  other  colloids,  namely  colloidal  ferric  hydrate  and  sodium 
oleate.  He  found  that  whatever  the  colloid  employed  the  rela- 
tive coagulating  power  of  the  various  salts  was  the  same.  Excep- 
tions to  this  rule  were  noted,  however,  when  salts  of  di-  or  tri- 
basic  acids  were  employed,  the  order  of  their  efficacy  in  coagu- 
lating egg-globulin  and  gelatin  being  the  same,  but  differing 


110 


CHEMICAL  STATICS 


when  ferric  hydrate  and  sodium  oleate  were  employed.  Nasse 
(49),  in  discussing  the  hypothesis  that  the  coagulation  of  proteins 
is  due  to  the  withdrawal  of  water  from  the  colloid,  had  raised  the 
objection  that  the  ratio  of  the  concentrations  at  which  the  mag- 
nesium and  ammonium  sulphates  bring  about  coagulation  of 
different  colloids  is  not  always  exactly  the  same.  For  different 
colloids  Nasse  found  the  ratio 

cone,  of  (NH4)2S04  just  sufficient  to  coagulate 
cone,  of  MgSO4  just  sufficient  to  coagulate 

possessed  the  following  values: 


Gelatin 

Egg-albumin 

Serum-albumin 

Hemi-albumose 

Peptone 

0.84 

1.03 

0.94 

0.85 

1.00 

Hofmeister,  however,  pointed  out  that  the  absolute  concen- 
trations which  are  required  to  bring  about  coagulation  in  these 
cases  are  very  different,  and  an  exact  quantitative  relation  of 
this  kind  could  not  be  expected  to  hold  good,  since  the  condition 
of  the  salts  in  solution,  or,  as  we  should  now  express  it,  their 
relative  degree  of  electrolytic  dissociation,  differs  at  different 
absolute  concentrations. 

Following  up  the  idea  that  the  coagulation  of  colloids  by  salts 
is  attributable  to  the  possession  by  the  salts,  in  the  concentra- 
tions employed,  of  a  greater  power  of  binding  water  than  that 
possessed  by  colloid,  Hofmeister  (1890-91)  took  up  the  study  of 
the  swelling  or  absorption  of  water  by  colloids  in  various  solutions, 
the  degree  of  swelling  in  different  solutions  being  regarded  as  a 
measure  of  the  relative  binding  capacities  of  the  colloid  and  of 
the  dissolved  salts.  He  found  that  in  solutions  of  sulphates,  tar- 
trates,  acetates,  alcohol,  cane-sugar  or  grape-sugar  gelatin-plates 
take  up  less  water  than  they  do  when  immersed  in  distilled 
water,  while  in  solutions  of  potassium,  sodium  or  ammonium 
chlorides,  sodium  chlorate,  sodium  nitrate  and  sodium  bromide 
they  take  up  more  water  than  they  do  when  immersed  in  distilled 
water.  It  will  be  recollected  that  the  sulphates,  tartrates  and 
acetates  are  the  most  energetic  coagulants  of  gelatin.  Regarding 
their  high  coagulating  power  as  being  attributable  to  their  power 
of  abstracting  water  from  the  protein,  the  interpretation  of  these 
results -of  Hofmeister's  becomes  clear. 


EARLIER  INVESTIGATIONS 


111 


In  1898  Pauli  (51)  (58)  published  the  results  of  a  number  of 
investigations  upon  the  influence  of  various  salts  on  the  gelatin- 
izing- and  melting-temperatures  of  gelatin  solutions.  He  found 
that  effects  of  different  salts  upon  the  gelatinizing  and  melting 
of  strong  gelatin  solutions  ran  parallel  with  their  power  of  coagu- 
lating gelatin  and  of  inhibiting  the  swelling  of  gelatin  plates. 
Thus  chlorides,  bromides,  and  iodides  of  potassium,  sodium,  am- 
monium and  magnesium  lower  the  temperatures  of  gelatinization 
and  of  melting,  in  the  following  order,  the  most  effective  being 
placed  first.* 

Sodium  sulphate,  magnesium  sulphate,  sodium  citrate,  ammo- 
nium sulphate,  magnesium  sulphate,  sodium  tartrate,  sodium 
acetate.  The  following  is  the  order  in  which  these  salts  bring 
about  the  coagulation  of  gelatin,  the  most  effective-  being,  placed 
first: 

Sodium  sulphate,  potassium  sulphate,  sodium  citrate,  mag- 
nesium chloride,  sodium  tartrate,  magnesium  sulphate,  ammonium 
sulphate,  sodium  acetate^  potassium  chloride,  sodium  chloride. 

There  is  evidently  a  close,  although  not  an  absolute  parallel- 
ism between  the  two  series.  Urea  and  alcohol  lowered,  but 
glycerin  markedly  raised  the  temperatures  of  gelatinization  and 
of  melting. 

In  a  later  communication  (52)  Pauli  showed  that  the  order  in 
which  various  salts  affect  the  coagulation-temperature  of  egg- 
globulin  is  similar  to  that  in  which  they  occur  in  the  above  series. 
The  following  table,  cited  after  Pauli,  gives  the  order  in  which 
the  various  salts  bring  about  the  transformation  of  the  proteins 
from  the  dissolved  into  the  solid  condition: 


Coagulation  of 
egg-globulin 
(Hofmeister) 

Coagulation  of 
gelatin 
(Pauli) 

Heat  coagulation 
of  egg-globulin 
(Pauli) 

Gelatinization  of 
gelatin 
(Pauli) 

Swelling  of  gela- 
tin 
(Hofmeister) 

Sulphates 
Acetates 
Citrates 
Tartrates 
Chlorides 
Chlorates 

Sulphates 
Citrates 
Tartrates 
Acetates 
Chlorides 

Chlorides 
Acetates 
Sulphates 
Chromates 
Chlorates 
Nitrates 

Sulphates 
Citrates 
Tartrates 
Acetates 
Chlorides 
Chlorates 

Sulphates 
Citrates 
Tartrates 
Acetates 
Chorides 
Chlorates 

Nitrates 

Bromides 

Nitrates 

Nitrates 

Iodides 

Bromides 

Bromides 

Iodides 

All  of  these  substances  were  employed  in  equimolecular  solutions. 


112  'CHEMICAL  STATICS 

The  nature  of  the  cation  was,  however,  found  to  be  not  wholly 
without  influence,  since  the  temperature  at  which  egg-globulin 
coagulates  is  lowest  in  the  presence  of  ammonium  chloride,  and 
highest  in  the  presence  of  magnesium  chloride,  the  series  being 
as  follows:  NH4,  K,  Na,  Li,  Ba,  Mg.  The  curves  expressing  the 
relationship  between  the  concentrations  of  the  salts  employed 
and  the  temperature  of  coagulation  are,  however,  not  parallel, 
and  in  some  cases  cut  one  another,  so  that  at  higher  concentra- 
tions (4  N)  the  series  runs  as  follows :  Na,  NH4,  Li,  Ba,  Mg. 

Pauli  also  investigated  the  effect  of  mixtures  of  two  salts,  and 
found,  as  had  already  been  indicated  by  the  results  of  Schafer 
(71),  that  the  coagulating  action  of  salts  upon  proteins  is  additive, 
that  is,  each  salt  exerts  its  separate  effect  and  the  precipitating 
(or  dissolving)  power  of  the  mixture  is  the  algebraic  sum  of  the  sepa- 
rate effects  exerted  by  its  components  except  when  the  two  salts 
have  a  common  ion  and  so  diminish  each  other's  degree  of  disso- 
ciation. This  result  has  since  been  more  fully  confirmed  by 
Pauli  (53)  and  by  Mellanby  (47). 

The  globulins,  as  a  class,  are  insoluble  in  distilled  water,  but 
are  soluble  in  dilute  saline  solutions.  Further  addition  of  salt 
results  in  coagulation  of  the  globulin.  Non-electrolytes  can  bring 
about  the  coagulation  but  not  the  solution  of  the  globulin.  From 
these  facts  Pauli  concluded  that  the  solution  of  globulin  is  due 
to  the  formation  of  compounds  of  the  globulin  with  salts,  of  the 

tyPe:  Na  -  globulin  -  Cl. 

the  further  addition  of  salts  to  this  solution  resulting  in  the 
precipitation  of  this  compound. 

3.  The  Influence  of  the  Electrical  Condition  of  the  Proteins 
upon  their  Precipitation  and  Coagulation  by  Electrolytes.  —  The 
action  of  very  small  quantities  of  salts  in  bringing  about  the 
precipitation  of  colloids  from  their  solution  was  first  studied  by 
Graham  (20).  He  ascertained,  for  example,  that  a  solution  of 
colloidal  aluminium  hydrate  prepared  by  dialysing  the  chloride 
is  so  sensitive  to  the  presence  of  salts  that  the  mere  addition  of 
a  few  drops  of  undistilled  water  suffices  to  precipitate  it.  This 
type  of  action  of  salts  upon  colloids  was  further  investigated  by 
Schultz  (73),  Prost  (66),  and  Linder  and  Picton  (44). 

Defining  the  precipitating-power  of  a  salt  as  the  reciprocal 
of  the  concentration  in  gram-molecules  per  litre  necessary  to 
coagulate  a  given  solution  of  the  hydrosol  of  sulphide  of  arsenic, 
Schultz  found  that  the  relative  precipitating-powers  of  the  uni- 


INFLUENCE  OF  ELECTRICAL  CONDITION 


113 


valent,  divalent  and  trivalent  metals  are  in  the  ratios  1:  30: 1650. 
Prost,  employing  the  hydrosol  of  cadmium  sulphide,  obtained  a 
similar  relation,  while  Linder  and  Picton  found  that  the  precipi- 
tating powers  of  different  salts  of  a  given  metal  are  proportional 
to  their  equivalent  conductivities  and  that  the  relative  precipi- 
tating powers  of  the  sulphates  of  univalent,  divalent,  and  trivalent 
metals  can  be  expressed  by  the  ratios  1 :  35 :  1023. 

In  all  of  these  cases  the  colloid  employed  was  electronegative, 
that  is,  on  electrolysis  it  migrated  to  the  anode.  The  experi- 
ments which  we  have  cited  show  that  in  such  cases  the  ion  of 
the  added  electrolyte  which  is  effective  in  bringing  about  precipi- 
tation is  the  cation.  In  1899,  however,  Hardy  (21)  showed  that 
egg-albumin,  modified  by  heating  its  solution  so  as  to  partially 
coagulate  the  protein,  may  be  induced  to  travel  either  to  the 
anode  or  to  the  cathode  by  simply  changing  the  reaction  of  its 
solutions.  In  acid  solutions  the  protein  behaves  like  a  cation, 
migrating  to  the  cathode;  in  alkaline  solutions  it  behaves  like 
an  anion,  migrating  to  the  anode.  In  alkaline  solutions,  the 
cations  of  added  salts  proved  to  be  the  active  agents  in  precipi- 
tating egg-albumin,  while  in  acid  solutions  the  precipitating 
power  of  the  cation  proved  to  be  altogether  subordinate  to  that 
of  the  anion  (22).  The  following  illustrate  his  results: 

PROTEIN  IN  PRESENCE  OF  TRACE  OF  ALKALI,  ELECTRO- 
NEGATIVE 
Temperature  16  degrees.     Coagulating  salt  1  gram-mol.  in  80,000  cc. 


Coagulated  at  once 

On  slightly  warming 

Did  not  coagulate 

A12(S04)3 
Cd(N03)2 
CuSO4- 
CuCl2 

MgS04 
BaCl2 
CaCl2 

Na2S04 
K2S04 
NaCl 

PROTEIN  IN  PRESENCE  OF  TRACE  OF  ACID,  ELECTRO- 
POSITIVE 


Coagulated  instantly 

No  effect 

A12(S04)3 
CaSO4 
K2S04 
Na2SO4 
MgSO4 

CuCl2 

Cd(N03)2 
BaCl2 
NaCl 

114  CHEMICAL  STATICS 

Similar  results  were  obtained  with  other  colloids.  Electroneg- 
ative colloids  are  precipitated,  if  at  all,  by  cations;  electropositive 
colloids,  by  anions. 

Whetham  (78)  (26)  explained  these  phenomena  in  the  following 
way:  He  assumes  that  at  the  surface  of  the  colloid  particles  there 
exists  a  double  electrical  layer.  When  the  particles  move  towards 
the  anode  they  are  negatively  charged,  when  they  move  to  the 
cathode  they  are  positively  charged.  The  surface  energy  of  the 
colloidal  phase  is  reduced  by  the  presence  of  the  electrical  double 
layer  and  therefore  its  tendency  to  contract.  The  existence  of 
the  double  layer,  therefore,  conduces  to  the  stability  of  a  system 
in  which  the  surface  of  the  colloid  is  greatly  extended,  i.e.,  to  the 
stability  of  the  "  colloidal  solution."  The  cations  of  the  added  elec- 
trolyte, in  the  case  of  electronegative  colloids,  or  the  anions  in  the 
case  of  electropositive  colloids,  neutralize  this  charge  and  therefore 
increase  the  energy  of  the  surface  of  the  colloidal  phase  and  its 
tendency  to  contract.  Hence  the  finely  suspended  colloidal  par- 
ticles unite  to  form  large  aggregates  having  a  less  extended  surface, 
and  these  aggregates  finally  become  so  large  as  to  assume  the 
properties  of  matter  in  mass,  and  hence  are  carried  out  of  solu- 
tion by  the  action  of  gravity.  In  this  way  the  dependence  of 
the  precipitating-power  of  an  electrolyte  upon  its  dissociation, 
found  by  Linder  and  Picton  and  by  Hardy,  and  also  the  reversal 
in  the  relative  precipitating-powers  of  the  ions  of  the  added 
electrolyte  upon  reversion  of  the  sign  of  the  electrical  charge 
carried  by  the  colloid,  found  an  explanation.  In  interpreting 
the  valency  rule  discovered  by  Schultz,  Whetham  develops  his 
theory  as  follows: 

"In  a  solution  where  ions  are  moving  freely,  the  probability 
that  an  ion  is  at  any  instant  within  reach  of  a  fixed  point  is, 
putting  certainty  equal  to  unity,  approximately  represented  by 
a  fraction  proportional  to  the  ratio  between  the  volume  occupied 
by  the  spheres  of  influence  of  the  ions  and  the  whole  volume  of 
the  solution,  and  may  be  written  as  AC,  where  A  is  a  constant 
and  C  represents  the  concentration  of  the  solution.  The  chance 
that  two  such  ions  should  be  present  together  is  the  product  of 
their  separate  chances,  -that  is  (AC)2.  Similarly  the  chance  for 
the  conjunction  of  three  ions  is  (AC)3,  and  for  the  conjunction 
of  n  ions  is  (AC)n." 

"In  order  that  three  solutions,  containing  trivalent,  divalent, 


INFLUENCE  OF  ELECTRICAL  CONDITION  115 

and  univalent  ions  respectively  should  have  equal  coagulative 
powers,  the  frequency  with  which  the  necessary  conjunctions 
should  occur  must  be  the  same  in  each  solution.  We  should 
then  have,  the  constant  being  assumed  equal  in  each  case, 

A2"C32n  =  A3"C23n  =  A6nd&n  =  a  constant  =  B. 


Therefore 


B~«.  B^  B^ 

T>  '"A'  X' 


Cij  Cz,  Ca,  representing  the  concentrations  of  monads,  diads., 
and  triads  in  their  respective  solutions.  Thus  we  get  for  the 
ratios  of  the  concentrations  of  equi-coagulative  solutions  : 


J_        _L        J 
BGn  :  B*n  :  B2 


_ 
Let  us  put  B6n  =  -',  the  ratios  can  then  be  written: 


The  reciprocals  of  the  numbers  expressing  the  relative  concen- 
trations of  equi-coagulative  solutions  give  values  proportional  to 
the  coagulative-powers  of  solutions  of  equal  concentration;  so 
that,  calling  the  coagulative-powers  of  equivalent  solutions  con- 
taining monovalent,  divalent,  and  trivalent  ions  respectively,  pi, 
P2,  PS,  we  get: 

Pi'.pz'.pa  =  l:x:x2, 

Let  us  now  take  some  numerical  examples. 
Putting  x  =  32  we  get  the  series: 

1:32:1024. 

Which  agrees  very  well  with  Linder  and  Picton's  results  for 
colloidal  solutions  of  antimony  sulphide: 

1:35:1023; 
and  putting  x  —  40,  we  get 

1:40:1600, 

numbers  comparable  to  Schultze's  values  for  sulphide  of  arsenic" 
(79). 


116  CHEMICAL  STATICS 

It  is  curious  that  during  the  discussions  which  immediately 
followed  these  discoveries  the  fact  should  have  been  so  generally 
overlooked  that  the  above  hypothesis  of  Whetham's  is  simply 
a  statement,  in  kinetic  terms,  of  the  Guldberg  and  Waage  mass- 
law.  According  to  this  law,  the  velocity  with  which  any  given 
reaction  proceeds  varies  directly  as  the  active  masses  of  each 
of  the  reacting  molecules.  In  the  case  under  consideration, 
presuming  that  a  given  number  (e.g.,  one)  of  molecules  of  protein 
react  with  one  molecule  of  a  salt  of  a  monovalent  metal  to  form 
a  compound,  then  twice  as  many  molecules  of  the  protein  may  be 
supposed  to  react  with  a  molecule  of  a  salt  of  a  divalent  metal, 
and  three  times  as  many  with  a  salt  of  a  trivalent  metal.  Assum- 
ing that  the  active  mass  of  the  colloid  (the  molecular  concentra- 
tion multiplied  by  the  degree  of  dissociation)  is  the  same  in  each 
of  these  cases  (which  is  also  assumed  in  Whetham's  theory)  and 
equal  to  A,  calling  the  initial  velocities  of  the  respective  reactions 
Vit  vz,  and  vs  and  the  concentrations  of  the  mono-,  di-  and  trivalent 
ions  Ci,  Czj  and  Cs,  we  have: 

Vi  is  proportional  to  Aci, 
Vz  is  proportional  to  A2Cz, 
03  is  proportional  to  A3c3, 

whence  it  follows  that  if  Vi  =  v2  =  VB  and  the  velocity-constants  of 
the  three  reactions  are  equal  (which  is  also  assumed  in  Whetham's 
hypothesis)  :* 


and—  ,  —  and  —  ,  i.e.,  the  dilutions  of  the  mono-,  di-  and  trivalent 

Ci  '  C2  C3  ' 

ions  at  which  combination  proceeds  with  equal  velocity  are  re- 
lated to  one  another  in  the  same  way.  Now  in  the  experiments 

described  above,  -  is  denned  as  p,  the  precipitating-power  of  the 

salt,  hence: 

Pi'.pz'.ps:  :  I:  A:  A2, 

which  is  exactly  the  relation  deduced  by  Whetham.  The  rela- 
tions found  by  Schultze,  Prost,  Linder  and  Picton  and  others, 
are,  therefore,  just  as  explicable  upon  the  assumption  that  the 

*  In  the  terminology  of  Whetham's  hypothesis,  the  term  "velocity  con- 
stant" would  read  "proportion  of  effective  collisions." 


INFLUENCE  OF  ELECTRICAL  CONDITION  117 

colloid  reacts  chemically  with  the  precipitating  ion  as  upon 
the  assumption  that  the  precipitating  ion  acts  merely  through 
altering  the  electrical  condition  of  the  colloidal  particles.  The 
former  view  attributes  to  the  colloids  no  especial  qualities  which 
differentiate  them  from  other  chemical  systems,  the  latter  view 
necessarily  involves  assumptions  regarding  the  nature  of  col- 
loidal solutions  which  have  hitherto  proved  incapable  of  veri- 
fication. 

It  is  not  surprising  that  velocities  rather  than  equilibria  deter- 
mine the  "precipitating-powers"  of  reagents  for  colloids,  when 
we  recollect,  firstly,  the  enormous  part  played  by  the  velocity  of 
change  in  the  final  physical  condition  of  a  colloid, f  and  secondly 
the  method  by  which  these  "precipitating-powers"  are  measured. 
Linder  and  Picton  measured  the  precipitating-power  of  a  salt  by 
titration,  running  the  solution  of  the  salt  into  the  solution  of 
arsenic  sulphide  until  coagulation  just  began  to  be  perceived. 
They  expressly  state  that  unless  the  time  occupied  in  the  titra- 
tion be  kept  approximately  the  same,  serious  deviations  from  the 
above  rule  occur,  "as  a  quantity  of  coagulant  insufficient  to  pro- 
duce coagulation  immediately  will  do  so  in  the  course  of  time." 
Under  these  conditions,  what  is  actually  measured  is  the  con- 
centration of  the  precipitating  agent  which  is  requisite  to  bring 
about  a  given  degree  of  change  (visible  precipitation)  within  a 
given  brief  period,  that  is,  a  velocity  and  not  an  equilibrium. 
Data  regarding  the  degree  of  precipitation  after  varying  periods 
and  the  equilibria  which  are  attained  in  such  reactions  as  these 
are  lacking,  but  Linder  and  Picton  have  shown  that  the  cation 
which  brings  about  the  precipitation  of  colloidal  arsenic  is  bound 
by  the  colloid  and  carried  down  with  it.  It  cannot  be  washed  out 
with  water,  but  it  can  be  replaced  by  another  metal.  Whitney 
and  Ober  (80)  obtained  similar  results  and,  moreover,  showed  that 
different  metals  are  carried  down  by  this  colloid  in  equivalent- 
molecular  proportions. 

That  these  results  are  susceptible  of  purely  chemical  inter- 
pretation was  pointed  out  by  Hardy  (22)  in  1900  and  this  view 
was  further  developed  by  him  in  his  communications  on  globulin 
referred  to  above  (24).  He  showed  that  in  a  series  of  salts  of 

*  According  to  W.  B.  Hardy  (23),  the  more  slowly  the  division  into  two 
phases  occurs  the  smaller  and  less  curved  is  the  surface  of  separation.  Cf. 
also  Freundlich  (15). 


118  CHEMICAL  STATICS 

the  same  valency  the  power  of  precipitating  an  electronegative 
colloid  varies  directly  as  the  equivalent  conductivity  of  the  salt, 
that  is,  as  the  active  mass  of  the  dissociated  metal  ions,  Since 
electronegative  colloids  appear  to  behave,  electrically,  like  the 
anions  of  an  acid,  and  electropositive  colloids  like  the  cations 
of  a  base,  their  respective  affinities  for  the  basic  and  acid  radicals 
of  salts  are  readily  explicable.  This  fact  appears,  however,  to 
have  been  first  clearly  pointed  out  by  Loeb  (45). 

An  interpretation  of  the  phenomena  attending  the  precipita- 
tion of  ionic  (" electrically  active")  colloids  by  electrolytes  prac- 
tically identical  with  that  of  Whetham  was  afterwards  brought 
forward  by  Bredig  (7). 

Modifications  of  Whetham's  theory  have  been  advanced  by 
Billitzer  (3)  (4)  and  by  Freundlich  (15)  (16).  Billitzer  objected 
to  the  assumption  which  was  made  by  Whetham  (although  as 
we  have  seen,  it  was  not  at  all  essential  to  his  hypothesis)  that 
an  electrical  double  layer  exists  at  the  surface  of  colloid  particles 
in  aqueous  solution  and  showed  that  this  assumption  is  inadequate 
to  explain  the  migration  of  the  particles  in  an  electrical  field. 
The  charge  on  the  colloid  particles  must  therefore  be  supplied 
by  the  particles  themselves,  by  giving  up  oppositely  charged 
ions  to  the  solution,  i.e.,  the  colloids  must  be  electrolytes.  So  far 
his  view  is  identical  with  that  which  is  developed  above.  But 
he  adds  to  this  the  assumption  that  the  charge  which  is  carried 
by  the  colloid  particles  is  not  a  full  atomic  charge.  The  precipi- 
tating action  of  oppositely  charged  ions  he  attributes  to  the 
electrostatic  attraction  of  the  colloid  particles  by  the  oppositely 
charged  crystalloid  ion.  Since  the  colloid  particle  is  assumed  not 
to  possess  a  full  atomic  charge,  a  number  of  particles  must,  he 
considers,  be  attracted  by  one  crystalloid  ion,  thus  forming  a 
molecular  group  large  enough  to  be  appreciably  acted  on  by 
gravity.  How  the  colloid  particles  come  to  acquire  a  fraction 
of  an  atomic  charge  Billitzer  does  not  explain,  nor  does  our  ex- 
perience of  the  behavior  of  electrolytes  afford  any  legitimate 
precedent  for  such  an  assumption. 

Freundlich  believes  that  the  precipitating  ion  must  penetrate 
(be  "adsorbed"  by)  the  surface  of  the  colloid  particle  and  that 
the  surfaces  of  colloids  are  permeable  to  ions  possessed  of  an 
opposite  but  not  to  ions  possessed  of  the  same  charge  as  their 
own.  The  precipitating  powers  of  various  salts  are  stated  to 


LATER  INVESTIGATIONS  119 

run  parallel  with  the  degree  to  which  they  are  "adsorbed"  by 
finely  pulverized  charcoal. 

Biltz  (5)  also  considers  that  the  precipitation  of  colloids  by 
electrolytes  and  by  other  colloids  is  due  to  the  formation  of 
"adsorption  compounds." 

4.  Later  Investigations  on  the  Significance  of  the  State  of 
Hydration  of  the  Proteins  in  Relation  to  their  Coagulation  by 
Electrolytes.  —  Continuing  his  investigations,  cited  above,  on  the 
coagulating  action  of  pairs  of  salts  on  egg-white,  Pauli  (53)  found 
that  a  number  of  salts  which  will  not  coagulate  egg-white  by 
themselves  will  increase  the  coagulating  power  of  other  salts 
when  mixed  with  them,  while  others  markedly  diminish  the 
coagulating  power  of  salts  which,  in  their  absence,  readily  coagu- 
late egg-albumin.  Moreover  certain  salts,  although  very  soluble, 
do  not  in  any  concentration  cause  coagulation  of  egg-white. 
The  possibility  suggested  itself  that  the  coagulating  action  of 
salts  might  depend  upon  two  antagonistic  factors,  respectively 
attributable  to  the  cations  and  the  anions  of  the  salts.  In  con- 
firmation of  this  view  it  was  found  that  the  NEU  ion,  when  com- 
bined with  S04  will  coagulate  egg-albumin,  although  when  com- 
bined with  acetanion  it  will  not.  Acetanion  coagulates  when 
combined  with  sodium,  but  not  when  combined  with  ammonium. 
Pursuing  this  line  of  reasoning  and  Investigation  Pauli  concluded 
that  if  the  coagulating  powers  of  a  series  of  cations  be  indicated 
by  /,  /',  /",  .  .  .  and  the  opposite  (solvent)  powers  of  a  series 
of  anions  by  h,  h',  h",  .  .  .  ,  then  in  a  mixture  of  electrolytes  the 
following  possibilities  exist : 

2  (/,/',  A  .  .  .)iz(M',ft",  •  •  0, 

the  mixture  being  such  as  to  coagulate,  leave  unaffected  or  in- 
hibit the  coagulation  of  the  albumin.  Pauli  found  that  in  egg- 
white  (in  which  the  protein  is  electronegative)  the  cations  of 
added  electrolytes  are  the  active  agents  in  inducing  coagulation, 
while  the  anions  inhibit  coagulation.  In  the  following  table  of 
Pauli's  the  cations  are  arranged  in  ascending  order  of  precipi- 
tating-power  from  left  to  right,  while  the  anions  are  arranged 
vertically,  the  weakest  inhibitor  coming  first  and  the  strongest 
last.  A  (+)  indicates  that  the  salt  which  results  from  the  union 
of  the  cation  and  anion  causes  coagulation  of  egg-albumin;  while 
a  (  — )  indicates  that  it  does  not. 


120 


CHEMICAL  STATICS 


Cations 

Mg 

NH4 

K 

Na 

Li 

Fluoride 

4- 

4. 

4. 

Sulphate  . 

4- 

4- 

4- 

4. 

4- 

Phosphate 

4- 

4- 

4- 

Citrate  .... 

4- 

4- 

4- 

Tartrate  

4- 

4- 

4- 

Acetate  

_ 

4- 

4- 

Chloride  

_ 

_ 

4- 

4- 

4- 

Nitrate  

_ 

_ 

4- 

4- 

Chlorate 

4- 

Bromide 

_ 

+ 

Iodide 

Thiocyanate  . 

_ 

_ 

_ 

_ 

The  valency  of  the  coagulating  ion  would  appear  to  be  quanti- 
tatively of  less  importance  in  the  coagulation  of  proteins  by 
salts  than  it  is  in  the  precipitation  of  ionic  protein  by  small 
quantities  of  salts,  since  magnesium  resembles  the  alkalies  closely 
in  its  coagulative  power,  while  lithium  approaches  the  alkaline 
earths. 

The  order  of  effectiveness  of  the  different  salts  in  bringing 
about  the  coagulation  of  electropositive  protein  is,  however,  exactly 
the  reverse  of  their  order  of  effectiveness  in  bringing  about  the 
coagulation  of  electronegative  protein,  such  as  the  albumin  in  egg- 
white.  This  was  first  shown  by  Posternak  (64),  who  employed 
the  reserve-material  of  the  seeds  of  Picea  excelsa,  dissolved  in 
very  dilute  hydrochloric  acid.  Posternak's  observation  has  been 
confirmed  by  Pauli  (53)  and  by  Hoeber  (29).  Acidifying  a 
solution  of  egg-white  reverses  the  functions  of  the  coagulating 
ions,  those  which  coagulated  electronegative  protein  most  strongly 
now  inhibit  its  coagulation  most  strongly.  Those  which  inhibited 
its  coagulation  now  induce  coagulation.  The  series  is  reversed 
in  every  respect;  the  anions  now  induce  coagulation  and  the 
cations  inhibit  it.  The  anions  precipitate  in  the  order: 

CNS  >  I  >  Br  >  NO3  >  Cl  >  Acetate, 
while  the  cations  inhibit  precipitation  in  the  order: 
Li  >  Na  >  K  >  NH4  >  Mg. 

When  electronegative  protein  (egg-white)  is  acted  upon  by 
salts  of  the  alkaline  earths  "irreversible"  precipitates  or  coagula 


LATER  INVESTIGATIONS  121 

are  formed,  insoluble,  that  is,  on  dilution  of  the  system  with 
water.  The  alkaline  earths,  moreover,  afford  a  strong  contrast 
to  the  alkalies,  in  that  the  precipitating-power  of  the  cation  is 
increased  by  the  anions,  in  the  series  which  is  characteristic  for 
electropositive  protein  (54).  In  other  words,  when  egg-white  is 
acted  upon  by  salts  of  the  alkaline  earths  the  protein  behaves,  so 
far  as  the  action  of  anions  upon  it  is  concerned,  as  though  it  were 
in  acid  solution.  Pauli  believes  that  the  alkaline  earth  reacts 
with  OH  groups  of  the  protein  to  form  the  comparatively  slightly 
dissociated  hydrates  of  the  alkaline  earths,  the  acid  which  is  set 
free  inducing  the  acid  reaction  of  the  medium.  In  other  words, 
Pauli's  view  is  that  protein  aids  the  action  of  water  in  bringing 
about  hydrolytic  dissociation.  Other  colloids  would  appear  to 
act  similarly,  since  Whitney  and  Ober  (80)  found  that  on  the 
addition  of  neutral  halogen  salts  of  the  alkaline  earths  to  col- 
loidal arsenic  sulphide  the  reaction  of  the  solution  becomes  acid. 
The  influence  of  added  salts  of  the  alkalies  and  magnesium 
upon  the  precipitation  of  proteins  by  heavy  metals  varies  with 
the  concentration  of  salt  employed  (55).  At  low  concentrations 
(0.005  m.)  the  salts  inhibit  precipitation  in  the  order: 

S04  <  Cl<  C2H302  <  N03  <  Br  <  I<  SON, 

while  in  high  concentrations  (4  m.)  they  encourage  precipitation 
in  the  order: 

SO4  >  Cl  >  C2H302  >  NO3  >  Br  >  I  >  SON. 

This  is  simply  a  particular  instance  of  the  rule,  to  which  the 
attention  of  the  reader  was  drawn  in  the  earlier  part  of  this 
chapter,  that  the  salts  may  act  as  precipitants  and  as  coagulants 
at  low  and  at  high  concentrations  respectively,  acting  as  solvents 
at  intermediate  concentrations.  The  heavy  metal  salts  afford  no 
exception  to  this  rule.  At  low  concentrations  they  precipitate,  at 
higher  concentrations  they  dissolve,  and  at  still  higher  concentra- 
tions they  coagulate  the  proteins  of  egg-white  (55).  Copper  sul- 
phate acting  upon  egg-albumin  would  appear  to  afford  an  excep- 
tion since  it  does  not  coagulate  even  in  saturated  solution.  That 
the  exception  is  apparent  and  not  real  is,  however,  shown  by  the 
observation  of  Galeotti  (17)  that  in  sufficiently  supersaturated 
solution  copper  albuminate  is  coagulated  by  copper  sulphate, 
addition  of  merely  saturated  copper  sulphate  solution  redissolves 


122  CHEMICAL  STATICS 

this  coagulum,  addition  of  water  then  results  in  the  precipitation 
of  copper  albuminate. 

The  concentration-range  throughout  which  the  salt  acts  as 
solvent  may  be  evanescent,  as  it  is  in  the  case  of  silver  nitrate 
acting  upon  egg-albumin  (Pauli). 

It  was  already  shown  in  1833  by  Rose  (68)  that  haemoglobin 
is  coagulated  by  concentrated  mercuric  chloride,  but  may  be 
redissoived  by  dilution,  to  be  precipitated  on  further  dilution. 

The  very  important  observation  has  been  made  by  Pauli  (56) 
that  absolutely  electrolyte  free  egg-albumin  is  not  ionic  (i.e., 
does  not  drift  in  an  electric  field)  and  that  under  these  conditions 
it  is  not  precipitable  by  heavy  metals.  It  is,  however,  coagulated 
by  highly  concentrated  salts.* 

According  to  Pauli  and  Handovsky  (57)  (60)  the  number  of 
ionized  protein  particles  in  a  solution  of  ionic  protein  is  dimin- 
ished by  the  addition  of  salts;  at  the  same  time  the  viscosity  of 
the  solution  diminishes  and  the  coagulability  of  the  protein  by 
the  usual  coagulating  agents  is  increased.  When  salts  are  added 
to  a  solution  of  electropositive  protein  (i.e.,  protein  combined 
with  acid)  an  increase  in  the  acidity  of  the  solution  results  (24). 
They  believe  that  acid-protein  reacts  with  salts  as  follows: 

H  H 

NH2(  NH2( 

/         XC1  /         XC1 

R(  +  NaN03  =  R\  +  HN03, 

XCOOH  XX)ONa 

while  alkali-protein  reacts  as  follows: 

/H  /K 

NH2(  NH2' 

/  OH  /         XC1 

R(  \     +  KC1  =  R\  +  H20. 

'XCOONa  NCOONa 

On  the  basis  of  the  more  probable  view  of  the  electrolytic 
dissociation  of  proteins  outlined  in  Chap.  I,  this  hypothesis 
would  be  represented  by  the  following  schematic  formulae: 

*  Egg-albumin  is  nearly  equally  basic  and  acid.  It  is  probable  that  the 
proteins  which  are  either  predominantly  acid  or  basic  may  still,  in  some 
measure,  be  electrolytically  dissociated  when  uncombined  with  bases  or  acids. 


APPLICATIONS  OF  THE  PHASE-RULE  123 

Cl  Cl 

^  I  ' 

-COH++  +  ^N-  +  NaN03  =  -CONa  =  N-  +  HN08 

I  I 

H  H 

OH  Cl 

-CONa++  +  ^N-  +  KC1  =  -CONa  =  N-'+  H2O. 
I  I 

H  K 

The  symmetry  of  the  action  of  the  salts  upon  the  acid  and 
alkali  protein  compounds  would  appear,  however,  to  indicate  a 
greater  similarity  in  the  structure  of  the  compounds  formed 
than  that  which  is  suggested  by  these  formulae. 

5.  Applications  of  the  Phase-rule  to  Protein-salt-water  Sys- 
tems. —  In  addition  to  the  above  investigations  attempts  have 
been  made  to  interpret  the  behavior  of  the  proteins  in  the  pres- 
ence of  salts,  in  the  light  of  the  phase-rule,  starting  from  the 
view  developed  by  Spiro  (74)  (10)  that  the  coagulation  of  pro- 
teins by  salts  might  be  regarded  as  a  separation  of  the  system 
into  two  phases,  a  solid  phase  rich  in  protein,  or  a  protein-salt 
compound,  and  poor  in  salt  and  water,  and  a  liquid  phase  poor 
in  protein,  rich  in  salt  and  water. 

The  phase-rule,  as  developed  by  Willard  Gibbs  (19),  van't 
Hoff  and  Roozeboom  may  be  enunciated  as  follows:  A  system  of 
r  coexistent  phases  containing  n  independently  variable  compo- 
nents is  capable  of  n  +  2  —  r  variations  in  the  temperature, 
total  pressure  of  the  system  or  concentration  of  the  components 
of  its  phases.  A  " phase"  is  a  portion  of  the  system  separated 
from  the  rest  by  a  definite  surface,  the  "  independently  variable 
components"  are  the  least  number  of  different  substances  with 
which  it  is  possible  to  represent,  in  a  chemical  equation,  the 
composition  of  each  phase  in  the  equilibrium  (14).  Only  equi- 
libria are  contemplated  in  the  derivation  of  the  phase-rule,  con- 
sequently it  is  not  applicable  to  systems  which  by  reason  of 
hysteresis  or  limited  reaction- velocity  *  are  in  a  condition  of 
incomplete  equilibrium.  A  discussion  of  the  theoretical  basis  of 

*  Unless  the  reaction-velocity  in  question  is  so  small  as  to  be  negligible  in 
comparison  with  the  velocity  with  which  the  equilibrium  under  examination 
is  attained;  for  example,  the  hydrolysis  of  protein  by  water  in  the  system 
protein-salt-water. 


124  CHEMICAL  STATICS 

the  phase-rule  would  be  out  of  place  here,  since  an  exposition 
of  the  principles  underlying  its  application  can  be  found  in  any 
general  work  on  physical  chemistry. 

Galeotti  (17)  (18)  has  applied  the  phase-rule  to  the  systems 
egg-albumin,  CuSO4,  H20,  serum-albumin  CuS04,  H2O;  serum- 
albumin,  AgN03,  H20;  egg-albumin,  AgN03,  H20;  while  Hardy 
(25)  has  applied  it  to  the  systems  serum-globulin,  salts,  H20. 
Both  observers  employ  the  triangular  co-ordinates  recommended 
by  Roozeboom,  in  which  the  three  sides  of  an  equilateral  triangle, 
each  equal  to  unity,  represent  the  components  of  the  system  and 
the  distances  of  a  point  P  from  these  axes,  measured  in  a  direc- 
tion parallel  with  the  sides  of  the  triangle,  express  the  compo- 
sition of  a  ternary  mixture  corresponding  to  the  point.  Since 
each  of  the  systems  investigated  by  these  observers  is  a  system 
consisting  of  two  immiscible  bodies,  protein  and  salt,  which  are 
both  partially  miscible  in  a  third,  water,  they  are  comparable 
with  the  system  water,  sodium  chloride  and  succinic  nitrile, 
which  has  been  studied  by  Schreinemakers  (72).  The  form  of 
the  isotherm  obtained  by  Hardy  and  by  Galeotti  is  exactly 
that  obtained  by  Schreinemakers  in  the  above-named  system. 

The  temperature  and  pressure  in  these  investigations  being 
arbitrarily  fixed  and  the  components  being  three  in  number  and 
in  equilibrium  with  water-vapor  at  the  pressure  and  temperature 
employed,  the  number  of  degrees  of  freedom  is  3  —  r,  where  r 
is  the  number  of  phases.  From  this  it  follows  that  for  a  given 
temperature  and  pressure  there  can  be,  in  general,  not  more  than 
three  phases  in  equilibrium  with  water-vapor  coexisting  in  the 
system  at  one  time. 

Now  Galeotti,  as  has  been  mentioned  above,  finds  that  solid 
CuSC>4  +  5  H20,  solid  protein  and  water  can  coexist  in  the  sys- 
tem at  the  same  time,  the  water  being  saturated  with  CuS04  + 
5  H2O  andralso  containing  some  dissolved  protein,  as  is  shown  by 
the  fact  that  dilution  of  the  fluid  phase  causes  further  precipitation 
of  protein.  From  this  it  follows  that  the  protein,  while  in  solution,  does 
not  constitute  a  separate  phase  and  is  not  divided  off  from  the  solvent 
by  a  surface  enwrapping  molecules  which  are  not  in  physical  con- 
tact with  the  fluid,  for  otherwise  there  would  be  four  coexistent 
phases;  this  simple  consideration  appears  to  have  been  over- 
looked by  the  majority  of  writers  on  colloids  and  by  many  of 
the  advocates  of  "adsorption." 


PRECIPITATION   AND  COAGULATION  125 

The  general  thermodynamic  conditions  of  equilibrium  in  sys- 
tems in  which  the  protein  or  other  colloid  forms  a  region  divided 
from  the  remainder  of  the  system  by  a  boundary  at  which  abrupt 
change  of  properties  takes  place  (as  in  a  suspended  coagulum 
or  a  jelly)  have  been  elaborated  by  Tolman  (75). 

In  his  first  communication  (17)  Galeotti  arrived  at  the  con- 
clusion that  proteins  do  not  form  unions  of  definite  composition 
with  copper,  since  the  copper  can  be  partially  and  progressively 
removed  from  the  protein  by  washing.  I  have  dwelt  in  a  pre- 
vious chapter  (Chap.  V)  upon  the  fallibility  of  this  reasoning. 
In  his  second  communication  (18),  however,  Galeotti  obtained 
data  which  indicate  that  at  the  moment  of  precipitation  of  egg- 
albumin  by  AgN03,  a  compound  is  formed  of  perfectly  definite 
molecular  weight  and  solubility  in  distilled  water.  These  data 
will  be  more  fully  discussed  in  the  succeeding  chapter. 

Keeping  the  quantities  AgN03  and  water  constant  and  alter- 
ing the  concentration  of  protein  (egg-albumin)  Galeotti  finds 
that  as  protein  is  successively  added  a  rapid  diminution  in  the 
number  of  silver  ions  occurs,  but  with  the  appearance  of  a  pre- 
cipitate the  diminution  proceeds  more  slowly  and  finally  tends  to  a 
constant  maximum. 

6.  The  Chemical  Mechanics  of  the  Precipitation  and  Coagu- 
lation of  Proteins  by  Salts.  —  We  have  seen  that  in  order  that 
precipitation  of  a  protein  by  salts  may  occur  the  protein  must 
be  ionized,  but  for  coagulation  this  condition  is  not  requisite. 
In  determining  the  rate  of  precipitation  the  valency  of  the  pre- 
cipitating ion  is  of  prime  importance,  in  determining  the  rate 
of  coagulation  it  is  of  comparatively  subordinate  importance. 
For  precipitation  very  low  concentrations  of  the  precipitating 
salt  suffice,  for  coagulation  high  concentrations  of  the  salt  are 
required.  This  latter  fact,  and  the  fact  that  the  presence  of 
coagulating  salts  aids  coagulation  by  alcohol  and  by  heat  suggests, 
as  it  did  to  Hofmeister,  that  coagulation  is  dependent  upon 
dehydration  of  the  protein. 

Starting  from  the  observation  of  Jones  and  Ota  (35)  that 
certain  salts,  when  dissolved  in  water,  produce  an  abnormal 
depression  of  the  freezing-point,  Jones  and  his  pupils  have  built 
up  a  very  large  body  of  evidence  for  the  existence  of  hydrates 
(or  "solvates")  of  substances  in  solution  (32)  (37).  These 
investigators  find  that  both  ions  and  undissociated  molecules  can 


126  CHEMICAL  STATICS 

form  "solvates,"  and  that  these  hydrates  or  "solvates"  are 
readily  decomposed  at  temperatures  which  approach  the  boiling 
point  of  the  solvent  and  by  the  presence  of  other  agents  in  the 
solution  which  compete  for  the  solvent.*  An  interesting  attempt 
has  been  made,  upon  the  basis  of  this  hypothesis,  to  explain  the 
color  changes  which  many  salts  undergo  in  the  presence  of  vary- 
ing amounts  of  water  or  of  dehydrating  agents  (37)  (33)  (36) 
(42)  (34).  As  is  well  known,  anhydrous  cobalt  chloride  is  blue, 
but  on  taking  up  water  it  becomes  violet  or  red.  Ostwald  (50) 
believed  that  the  undissociated  cobalt  chloride  is  blue,  while  the 
cobalt  ion  is  red.  Since,  however,  the  color  of  a  concentrated 
solution  of  cobalt  chloride  can  be  changed  from  purplish  red  to 
blue  by  the  addition  of  small  amounts  of  calcium,  or  still  smaller 
amounts  of  aluminium  chlorides,  or  by  the  addition  of  a  few 
drops  of  alcohol  (1)  Lewis  concludes  that  this  change  in  color 
is  due  to  dehydration  of  the  cobalt  chloride  molecule  in  solution, 
by  the  abstraction  of  water  from  it  by  the  added  substance, 
similar  conclusions  had  previously  been  reached  by  other  observ- 
ers (70)  (63)  (81)  (40)  (27)  (28).  Similarly  the  progressive 
change  in  color  of  cupric  chloride  solutions,  from  blue  to  greenish 
brown,  on  concentration  or  dehydration  is  attributed  to  the  loss 
of  water  on  the  part  of  cupric  chloride-water  complexes.  Lewis 
finds  that  if  various  bromides  be  added  to  concentrated  solutions 
of  cupric  bromide  the  copper  salt  is  dehydrated  (turned  brown) 
by  the  salts  of  monovalent  metals  in  the  order  Li  >  (Na,  NH4)  >  K. 
For  the  chlorides  the  order  was  Li  >  Na  >  NH4  >  K.  Di- 
valent metals  dehydrate  more  strongly,  the  order  being  Mg  > 

*  A  very  striking  experiment  illustrating  the  formation  of  "solvates"  is 
that  cited  by  Pickering  (6).  If  a  mixture  of  propyl  alcohol  and  water  be 
placed  in  a  semipermeable  vessel  and  surrounded  with  water,  it  is  found  that 
water  enters  the  cell,  but  that  no  propyl  alcohol  escapes.  If,  however,  the 
same  semipermeable  vessel,  containing  the  same  mixture  of  alcohol  and  water 
be  immersed  in  propyl  alcohol,  propyl  alcohol  enters  the  cell  and  water  does 
not  leave  it.  In  other  words  the  vessel  is  permeable  to  either  propyl  alcohol 
or  water  when  these  are  pure,  but  it  is  impermeable  to  mixtures  of  the  two, 
the  inference  being  that  large  molecular  complexes  are  formed  on  mixing  these 
reagents,  which  cannot  pass  through  the  pores  of  the  vessel.  From  these  and 
similar  experiments  Poynting  (65)  concludes  that  osmotic  pressure  is  an  ex- 
pression of  the  diminution  in  tKe  active  mass  of  the  solvent  due  to  the  formation 
of  compounds  with  the  dissolved  substance.  For  a  fuller  discussion  of  these 
and  similar  hypotheses  the  reader  is  referred  to  current  works  on  physical 
chemistry. 


PRECIPITATION  AND  COAGULATION  127 

Ca  >  Sr  >  Ba  while  trivalent  metals  (Al)  act  still  more  ener- 
getically. In  opposition  to  this  view  Donnan  (11)  (12)  (41)  (48) 
(13)  advances  the  hypothesis  that  the  blue  color  of  concen- 
trated solutions  of  cobalt  chloride  is  due  to  the  formation  of 
complex  ions  of  the  type  CoCl2.Cl2".  Lewis  points  out,  however, 
that  this  view  is  inconsistent  with  the  fact  that  it  is  possible  to 
change  the  color  of  the  solution  from  blue  to  red  by  mere  dilu- 
tion, without  altering  the  active  mass  of  any  component  of  the 
system  except  water.  Other  objections  against  Donnan's  view 
have  been  urged  by  Hartley  and  H.  C.  Jones. 

The  peculiar  interest  to  the  biological  chemist  of  the  possi- 
bility thus  indicated,  that  substances  dissolved  in  water  form 
loose  combinations  with  the  solvent,  lies  in  the  especial  signifi- 
cance of  water  in  relation  to  the  protein  and  polypeptid  struc- 
ture. As  indicated  in  the  first  chapter,  dehydration  of  a  protein 
may  result  in  the  following  reactions: 

/NH3OH        HOOC.  /NH3.OOC.R,NH3OH 

R;          +          /R  =  Rv  +  H2° 

NCOOH        HOH3NX  NCOOH 

,  NH3OOC.R.NH3OH         ,  NH.OC.R.NH3OH 
R'  =  R(  +  H20 

NCOOH  XCOOH 

,  NH.OC.R.NH3OH          ,  NH.OC.R.NH2 
R'  =  R'  +  H20 

XCOOH  XCOOH 

,  NH.OC.R.NH2          /  NH.OC.R.NH 
R'  =  R'  I    +H20 

XCOOH  XCO- 

and  hydration,  of  course,  may  result  in  the  reversion  of  this 
series  of  changes. 

That  proteins  may  be  thrown  out  of  solution  in  two  very  dif- 
ferent conditions  of  hydration  is  evident  from  the  researches 
cited  in  the  previous  sections  of  this  chapter;  it  is  even  more 
clearly  shown  by  the  following  experiments  (67): 

Anhydrous  casein  dissolves  readily  in  cold  anhydrous  *  formic 
acid;  still  more  readily  in  hot  formic  acid  If,  to  a  two  per  cent 
solution  of  casein  in  formic  acid,  we  add  a  fairly  concentrated 

*  Anhydrous,  that  is,  save  for  traces  of  moisture  derived  from  the  atmos- 
phere. 


128  CHEMICAL  STATICS 

solution  of  cupric  chloride,  the  mixture  is  at  first  green,  indicating 
the  presence  of  lower  hydrates  of  cupric  chloride,  but  on  adding 
more  of  the  solution  it  becomes  blue,  and  simultaneously  with 
the  appearance  of  a  pure  blue  color,  but  not  before,  precipitation 
of  cupric  caseinate  occurs.  If,  to  5  cc.  of  a  2  per  cent  solution  in 
formic  acid,  we  add  1J,  2,  or  2\  cc.  of  a  saturated  solution  of 
cupric  chloride,  no  precipitation  of  the  caseinate  occurs,  but  on 
diluting  this  mixture  with  water  a  precipitate  results,  and  the 
appearance  of  this  precipitate  coincides  with  the  attainment  of  a 
clear  blue  color  on  the  part  of  the  mixture. 

About  six  cubic  centimetres  of  water  are  required  to  produce 
a  permanent  precipitate.  This  precipitate  redissolves  on  heat- 
ing and  the  mixture  simultaneously  becomes  green;  on  cooling  the 
blue  color  reappears  and  with  it  the  precipitate.  If  formic 
acid  be  added  to  the  mixture  the  precipitate  redissolves  as  soon 
as  the  mixture  becomes  green.  If  the  precipitate  be  very  slight 
it  will  redissolve  on  adding  alcohol.  It  cannot  be  urged  that  the 
formation  of  cupric  caseinate  requires  the  presence  of  a  sufficient 
concentration  of  cupric  ions,  because  green  solutions  of  cupric 
chloride  contain  an  abundance  of  ions  *  and  casein  will  react 
with  very  small  amounts  of  metal  ions,  for  although  it  is  itself 
insoluble  it  will  drive  carbonic  acid  out  of  the  sparingly  soluble 
calcium  carbonate  to  form  a  freely  soluble  caseinate  of  calcium 
(vide  Chap.  V). 

If  instead  of  adding  water  to  a  mixture  of  5  cc.  of  2  per  cent 
casein  in  formic  acid  and  2  cc.  of  saturated  CuCU,  we  add  alcohol, 
no  coagulation  occurs  until  the  mixture  changes  in  color  from 
green  to  brown  when  a  coagulum  of  cupric  caseinate  is  produced 
which  redissolves  on  adding  water. 

Similar  results  are  obtained  when  a  2  m.  solution  of  cobalt 
chloride  is  employed  instead  of  a  saturated  solution  of  cupric 
chloride.  If  to  5  cc.  of  a  2  per  cent  solution  of  casein  in  formic 
acid  we  add  2  to  3  cc.  CoCl2  we  obtain  a  blue-purple  solution; 

*  Green  solutions  containing,  probably,  a  mixture  of  the  anhydrous  brown 
salt  and  the  fully  hydrated  blue  salt.  Even  on  the  basis  of  the  hypothesis 
urged  by  Donnan  (11),  (12),  therefore,  a  considerable  number  of  cupric  ions 
must  exist  in  green  solutions.  It  is  important  to  notice  that  the  precipitation, 
as  stated  above,  does  not  occur  until  the  solution  is  pure  blue  in  color;  mix- 
tures so  slightly  green  that  they  appear  wholly  blue  until  viewed  alongside 
pure  blue  mixtures  produce  no  precipitate. 


PRECIPITATION  AND  COAGULATION  129 

on  adding  water  to  this  mixture  it  changes  in  color  from  blue- 
purple  through  red-purple  to  clear  pink.  Not  until  a  pure  pink 
color  is  obtained  does  a  precipitate  result.  If,  instead  of  add- 
ing water,  we  add  a  considerable  volume  of  alcohol.  (10  volumes) 
the  mixture  rather  abruptly  changes  to  a  clear  pale  blue  and 
then,  but  not  before,  we  obtain  a  coagulwn  of  cobalt  caseinate. 

Electronegative  casein  is  not  precipitated  by  the  salts  of  the 
alkalies,  though  it  is  readily  precipitated  by  salts  of  the  alkaline 
earths.  Electropositive  casein  (i.e.,  casein  dissolved  in  acids)  is, 
however,  very  readily  precipitated  by  salts  and  these  precipitates 
are  not  soluble  upon  dilution.  Thus  if  2  cc.  of  N/1Q  HC1  be 
added  to  5  cc.  of  a  1  per  cent  solution  of  casein  in  0.008  N  KOH, 
a  clear  acid  solution  of  casein  results.  The  casein  is  precipitated 
from  this  by  the  addition  of  four  drops  of  a  saturated  solution 
of  sodium  chloride,  or  by  one  drop  of  a  saturated  solution  of 
ammonium  sulphate;  this  latter  precipitate  does  not  dissolve 
on  diluting  the  mixture  to  one-sixteenth. 

Casein  formate  is  no  exception  to  the  other  salts  of  casein  with 
acids,  but  the  precipitation  will  only  occur  in  the  presence  of  a 
sufficiency  of  water.  If  to  5  cc.  of  a  2  per  cent  solution  of  casein 
in  formic  acid  we  add  a  saturated  solution  of  ammonium  sulphate, 
3  cc.  of  this  solution  just  suffice  to  produce  a  coagulum;  this 
becomes  more  abundant  on  adding  water,  and  redissolves  on 
adding  formic  acid.  If,  however,  instead  of  adding  3,  we  add 
2  cc.  of  the  saturated  ammonium  sulphate  solution,  a  clear  solu- 
tion is  obtained.  On  adding  water  to  this  a  precipitate  results 
which  redissolves  on  heating  and  reappears  on  cooling. 

Analogous  results  may  be  obtained  with  ovomucoid. 

It  is  clear,  therefore,  that  protein  may  be  thrown  out  of  solu- 
tion by  electrolytes  in  two  grades  of  hydration,  the  one  of  high, 
the  other  of  very  low  hydration.  The  former  process  is  what 
we  have  termed  precipitation,  the  latter  we  have  defined  as 
coagulation.  At  grades  of  hydration  intermediate  between  these 
extremes  the  protein  may  be  soluble.  Dehydration,  partial  or 
complete,  leading  to  resolution  or  to  coagulation,  may  be  induced 
by  heat,  by  non-electrolytes  possessing  an  affinity  for  water  or 
by  electrolytes.* 

The  importance  of  a  high  degree  of  dehydration  in  the  pro- 

*  See  also  Chick  and  Martin  (10). 


130  CHEMICAL  STATICS 

duction  of  coagula  irresistibly  suggests  that  this  phenomenon  is 
dependent  upon  the  formation  of  anhydrides  *  analogous  to  the 
diketopiperazines  (vide  Chap.  I)  and  of  the  general  formula: 

/NH  /NH.OC, 

"R          I  nr       "R  T? 

XCO  XCO.HNX 

Such  bodies  may  exist  either  in  the  keto  form,  illustrated  by 
the  above  formulae,  or  in  the  enol  form,  such  as: 

,N.(HO)CX 
RxC(OH).Nx 

If  this  be  granted  then  the  fact  that  alcohol  throws  down  the 
protein  salts  in  an  unaltered  condition  (vide  Chap.  IV)  lends 
strong  support  to  the  view  which  I  have  advanced  (Chap.  I) 
regarding  the  mode  of  formation  and  structure  of  the  protein 
salts,  since  according  to  that  view  the  metal  ions  in  a  protein- 
metal  compound  would  be  bound  up  in  —  COH.N—  groups  pre- 
viously to  dehydration,  which  could  only  affect  terminal  —  NH2 
or  —  NH3OH  and  —  COOH  groups  and  would  leave  the  union  be- 
tween the  protein  and  the  metal  unaffected. 

In  this  connection,  and  with  reference  to  the  "solvate77  theory 
of  Jones,  it  is  of  interest  to  note  that  the  relative  efficacy  of  the 
various  salts  in  bringing  about  the  coagulation  of  electronegative 
protein  is  exactly  their  relative  efficacy  in  bringing  about  dimi- 
nution of  the  solubility  of  phenylthiocarbamid  (69)  (53).  On  the 
other  hand,  evidence  of  the  affinity  of  proteins  for  water  is  afforded 
by  the  observation  of  Pauli  and  Samec  (59)  that  the  solubility 
of  highly  soluble  inorganic  salts,  such  as  ammonium  chloride, 
magnesium  chloride  or  ammonium  sulphocyanate  in  water  is 
diminished  by  the  presence  of  gelatin  or  of  blood-serum  proteins. 
The  solubility  of  very  slightly  soluble  substances  (calcium  sul- 
phate, phosphate,  or  carbonate  and  uric  acid)  is,  however,  defi- 
nitely increased. 

As  regards  the  precipitation  of  proteins  by  salts;  it  appears 
probable  that  acid  and  alkali  protein  react  with  salts  as  follows: 

*  Cf.  also  Gustav  Mann  (46). 


PRECIPITATION  AND  COAGULATION  131 

H 

H2N.R.COH++  +  ^N.KCOOH  +  2  NaN08 
I 
Cl 

Na 
I 

=  H2N.R.CONa  =  N.R.COOH  +  2  HNO3       (i) 
I 

Cl 
H 

H2N.R.CONa++  +  ^N.R.COOH  +  NaCl 

I 
OH 

Na 
I 

=  H2N.R.CONa  =  N.R.COOH  +  H2O  (ii) 

I 
Cl 

Since  proteins,  dissolved  in  salt  solutions,  are  electrically  neutral 
(Hardy),  it  appears  probable  that  this  compound  undergoes 
internal  neutralization  thus  (vide  Chap.  I) : 

Na  Na 

I  I 

H2N.R.CONa  =  N.R.COOH  =  H3N.R.CONa  =  N.R.COO     (iii) 

I 


Cl 


Cl 


It  will  be  observed  that  this  hypothesis  is  a  slight  modification 
of  that  advanced  by  Pauli  and  Handovsky  (57)  in  that,  in  the 
first  place,  cognizance  is  taken  of  the  fact  that  the  proteins  ionize, 
not  at  terminal  —  NH2  or  —  COOH  groups  but  at  internal  enol 
groups  and,  in  the  second  place,  the  compounds  which  are  formed 
with  acid-  and  alkali-protein  respectively  are  symmetrical  in 
structure,  so  that  the  symmetry  of  the  effects  of  salts  in  dissolv- 
ing and  coagulating  these  compounds,  which  has  been  observed 
by  Pauli,  is  readily  accounted  for. 

In  dilute  solutions,  that  is,  solutions  in  which  the  active  mass 
of  water  is  great,  these  compounds  undergo  hydrolytic  dissocia- 
tion in  the  following  way: 


132  CHEMICAL  STATICS 

Na 
I 

H2N.R.CONa=N.R.COOH  +  H2O 
I 
Cl 

H 
I 

=  H2N.R.CONa  =  N.R.COOH  +  NaOH.       (iv) 
I 
Cl 

This  decomposition  will,  naturally,  take  place  more  readily 
in  dilute  acid  than  in  alkaline  solution;  more  concentrated  acid 
would,  of  course,  abstract  the  sodium  from  the  compound  and 
convert  the  whole  of  it  into  the  acid-protein  compound.  In 
either  case  the  neutral  compound  which  results  may  be  insoluble. 
When  the  active  mass  of  water  is  diminished,  however,  for  ex- 
ample by  the  addition  of  a  dehydrating  agent  or  of  a  salt  with 
an  affinity  for  water,  this  hydrolytic  decomposition  is  prevented, 
and  the  complex  salt: 

Na 
I 
HsN.R.CONa  =  N.R.COO 

I Cl  I 

may  pass  into  solution. 

Further  dehydration  leads  to  the  loss  of  —  H  and  —OH  by 
terminal  —  NH2  and  —  COOH  groups,  as  depicted  above,  and  the 
formation  of  complex  insoluble  anhydrides. 

This  hypothesis  furnishes  an  explanation  of  the  following  facts: 

(i)  That  the  addition  of  small  concentrations  of  neutral  salts 
to  a  solution  of  acid-protein  increases  the  acidity  of  the  solution, 
while  the  addition  of  salts  to  alkali-protein  solutions  does  not 
increase  their  alkalinity  (vide  equations  i  and  ii). 

(ii)  That  acid-protein  is  precipitated  by  cations,  alkali-protein 
by  anions  (vide  equation  iii). 

(iii)  Since  the  union  between  the  salt  and  the  protein  is  chemi- 
cal in  character,  Schultzes'  valency-rule  would  apply  to  the  rate 
of  precipitation  (Cf.  section  3). 

(iv)  The  reaction  (acidity  or  alkalinity)  of  the  system  being 
maintained  constant,  the  precipitation  of  the  protein  depends 
only  upon  the  active  mass  of  water  and  not  upon  the  active 


PRECIPITATION   AND  COAGULATION  133 

mass  of  salt,  provided  this  is  sufficient  to  enter  into  combination 
with  the  protein  (vide  equation  iii),  i.e.,  it  is  possible  to  bring 
about  precipitation  by  mere  dilution,  the  relative  masses  of 
protein  and  salt  being  unaltered. 

(v)  The  precipitation  of  proteins  by  salts  occurs  more  readily 
in  acid  than  in  alkaline  solutions  (vide  equation  iii). 

(vi)  The  observation  of  Bonamartini  and  Lombardi  (6)*  that 
egg-albumin  in  neutral  solution  combines  with  both  the  basic 
and  the  acid  radicals  of  copper  sulphate  in  equivalent  propor- 
tions to  form  an  insoluble  compound  but  that  in  alkaline  solu- 
tion it  combines  with  excess  of  copper  to  form  a  soluble  compound 
(vide  equation  iv).  In  alkaline  solution  hydrolytic  dissociation 
of  the  complex  salt  is  pushed  back  in  accordance  with  the  fol- 
lowing equations. 

CuS04  +  2KOH  -»  K2SO4  +  Cu(OH)2 
H2N.R.CO.N.R.COOH    H2N.R.CO.N.R.COOH 


/  '  \ 

Cu(OH)2-f        Cu(    S04;l         -»         Cu(    SO  )Cu       +2H2O 

\  i  / 


H2N.R.CO.N.R.COOH     H2N.R.CO.N.R.COOH 

(vii)  The  observation  of  Osborne  (Cf.  Chap.  V)  that  edestin 
crystallized  from  concentrated  salt-solutions  will  decompose  the 
salt,  binding  the  base,  is  probably  attributable  to  the  reaction: 

H 

H2N.R.CONa.N.R.COOH  +  NaCl 
I 
Cl  Na 

I 

=  H2N.R.CONaN.R.COOH  +  HC1 

I 

Cl 

the  complex  salt  being  in  this  case  insoluble. 

(viii)  The  observation  of  Pauli  that  precipitation  of  a  protein 
by  salts  when  it  is  non-ionic  is  impossible.  For,  when  the  pro- 
tein is  not  ionized  the  nitrogen  is  bound  up  in  undissociated 
—  COH.N—  groups  and  is  not  attached  to  H+  and  OH'  groups 
replaceable  by  the  ions  of  the  salt. 

*  The  egg-albumin  employed  by  these  observers  is  not  ash  free;  it  must, 
therefore,  according  to  Pauli  have  been  ionic. 


134  CHEMICAL  STATICS 

This  fact  also  explains  the  observation  of  Bugarszky  and  Lieber- 
mann  (8)  that  uncombined  and,  presumably,  unionized  proteins 
do  not  combine  with  salts  in  neutral  solutions.  These  observers 
used  egg-albumin,  which,  according  to  Pauli,  is  electrically  neutral 
when  uncombined  with  acids  or  bases. 

(ix)  The  observation  of  Hardy  and  Pauli  that  coagulation  of  a 
protein  by  salts  is  possible  whether  it  be  ionic  or  not,  since  the 
dehydration  of  terminal  —  NH2  and  —  COOH  groups  does  not 
depend  upon  the  dissociation  of  —  COH.N—  groups. 

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CHAPTER  VII 
THE   COMPOUNDS   OF  THE  PROTEINS    (Continued) 

1.  Compounds  with  the  Heavy  Metals.  —  From  what  has 
been  said  in  the  previous  chapter  concerning  the  mechanism  of  the 
precipitation  and  coagulation  of  proteins  by  salts,  it  will  be  clear 
that  the  percentage  of  a  heavy  metal  which  is  bound  by  protein 
must  depend  very  intimately  upon  the  mode  of  preparation  of  the 
compound.  For  instance,  in  the  presence  of  silver  nitrate  under 
conditions  involving  moderate  hydration  (e.g.,  a  fairly  high  con- 
centration of  the  silver  nitrate  or  the  presence  of  some  other 
dehydrating  agent),  or  in  alkaline  solution,  the  following  reaction 
may  occur:  jj 

I 
H2N.R.CONa.N.R.COOH  +  2  AgNO3 

I  Ag 

OH  I 

=  H2N.R.COAg.N.R.COOH  +  NaNO3 
I 
N03 

in  an  acid  medium,  or  under  conditions  involving  less  hydration, 
this  compound  could  not  exist  and  the  reaction 

Ag 

I 
H2N.R.COAg.N.R.GOOH  +  H2O 

I  H 

N03  I 

^H2N.R.COAg.N.R.COOH+  AgOH 
I 
N03 

must  occur.  In  a  still  more  acid  medium  the  silver  would  be 
altogether  abstracted  from  this  compound,  while  in  a  fairly 
strongly  alkaline  medium  the  compound 

H 

I 
H2N.R.COAg.N.R.COOH 

I 

OH 
137 


138  CHEMICAL  STATICS 

would  doubtless  be  formed.  In  the  majority  of  investigations 
upon  this  subject  no  care  has  been  taken  to  maintain  a  neutral  or 
otherwise  constant  reaction  in  the  medium  from  which  the  protein- 
heavy  metal  compound  is  thrown  out,  each  observer  has  prepared 
these  compounds  under  different  conditions,  and  no  clear  dis- 
tinction has  been  made  between  the  precipitation  of  the  protein  by 
the  heavy  metal  salt  and  its  coagulation.  It  is  not  matter  either 
for  surprise  or  bewilderment,  therefore,  that  different  observers 
have  obtained  very  conflicting  results  in  determining  the  heavy 
metal  content  of  these  compounds.  In  the  light  of  our  present 
knowledge  the  majority  of  these  results  are  perceived  to  be  mean- 
ingless, since  they  were  obtained  with  compounds,  or  mixtures  of 
different  compounds,  prepared  under  inadequately  controlled 
conditions.* 

From  the  observations  of  Galeotti  (30),  we  may  infer  that  at 
the  moment  when  precipitation  begins,  the  compound  of  egg-albumin 
with  silver  (or  silver  nitrate)  is  of  constant  molecular  weight  and 
solubility  in  water.  This  investigator  has  applied  to  the  system 
protein,  egg-albumin,  water,  the  law  of  Jahn,  that  the  influence 
which  an  electrolyte  exerts  upon  the  solubility  of  another  substance 
(its  dehydrating  or  hydrating  action)  is  a  function  of  three  vari- 
ables, namely,  the  concentration  of  the  undissociated  molecule  of 
the  electrolyte,  that  of  its  cation,  and  that  of  its  anion.  Gal- 
eotti measured  the  percentage  of  protein  precipitated  and  also  the 
concentration  of  silver  ions,  determining  the  latter  directly  with 
the  aid  of  a  concentration-chain  (potentiometer).  Calling  C  the 
molecular  concentration  of  a  saturated  solution  of  the  protein- 
silver-nitrate,  and  C'  its  solubility  in  the  presence  of  silver  nitrate, 
applying  Jahn's  law  we  have 


lognat.         =  [CotK  +  Ck  fou  + 

in  which  R  is  the  gas-constant;  <foi  (at  constant  temperature  and 
pressure)  is  the  constant  ratio  between  the  concentration  of  the 
non-dissociated  molecule  of  AgNOs  and  its  effect  upon  the  solu- 
bility of  the  protein-compound;  $n  and  <fc»i  are  similar  factors  for 
the  cation  and  the  anion  of  the  silver  nitrate;  and  C0  and  Ck  are 
the  concentrations,  respectively,  of  the  undissociated  and  the 

*  For  a  review  of  the  older  literature  upon  the  heavy  metal  compounds  of 
proteins,  Cf.  O.  Cohnheim  (25). 


HEAVY  METALS 


139 


dissociated  AgNO3,  the  concentration  of  the  cation  being  assumed 
to  be  equal  to  that  of  the  anion. 

Calling  Cg  the  total  concentration  of  silver  nitrate,  then  C0  = 
Cg  —  Ck,  and  we  have 


=          [CW>0i  +  Ck  fo 


where  C  is  the  molecular  concentration  of  the  protein-silver-nitrate 
in  saturated  watery  solution  (a  quantity  which  naturally,  cannot 
be  measured  directly)  and  C'  is  the  molecular  concentration  of  the 
protein-silver-nitrate  in  the  AgN03  solution. 

Assuming  that  the  molecular  weight  of  the  compound,  in  saturated 
solution,  is  always  the  same,  whether  excess  of  silver  nitrate  be  present 
or  not,  .we  can  take  C'  as  the  percentage  concentration  of  the  com- 

Ql 

pound,  since  the  ratio  ^-  will  then  not  involve  the  molecular  weight 

of  the  compound. 
Putting 

1  2  m     <f>Qi  2m 

~logC"a;   IT'logC"^'    "R" 

we  have,  from  the  above  equation, 
alogC'  +  0(V 


logC 


1=0, 

in  which  a,  0,  and  7  are  constants,  and  C1 ',  Cg,  and  Ck  are  all  directly 
measurable.  The  following  results  of  Galeotti's  demonstrate  the 
validity  of  this  equation,  the  most  probable  values  of  the  constants 
(a  =  -1.507;  0  =  +37.27;  7  =  -72.06)  having  been  determined 
from  all  the  observations  by  least  squares. 


«  X  log  C' 

fiXC, 

,xck 

Sum 

-0.207 
-0.136 
+0.140 
+0.501 
+0.741 

+0.933 
+1.465 
+1.759 
+2.250 
+3.063 

-1.741 
-2.341 
-2.872 
-3.741 
-4.813 

-1.015 
-1.012 
-0.973 
-0.990 
-1.009 

Averace 

-1.000 

From  this  result  we^may  draw  the  following  conclusions: 
(a)   The  substance  precipitated  is  a  compound  and  not  the  free 
protein,  since  the  value  of  a  in  the  above  equation  indicates  a 


140  CHEMICAL  STATICS 

limited  solubility  in  water  and  free  egg-albumin  is  miscible  with 
water  in  all  proportions.* 

(b)  This  compound  is  of  the  same  molecular  weight  in  all  of  the 

C' 

solutions  investigated,   otherwise  the  ratio  -~-  would  include   a 

0 

factor  expressing  the  relation  between  the  molecular  weight  of  the 
compound  and  the  excess  of  AgN03  in  its  saturated  solution.  Also 
the  constancy  of  C  (i.e.,  the  constancy  of  a)  shows  that  it  is  always 
the  same  substance  that  is  present  "in  solution  at  the  moment 
preceding  precipitation. 

(c)  This  compound  is  probably  not  ionized,  since  Jahn's  law 
applies  only  to  the  influence  of  electrolytes  upon  the  solubility  of 
non-electrolytes . 

(d)  The  quantity  of  NO3'  bound  by  the  protein  just  before 
precipitation  must  be  the  same  as  the  quantity  of  Ag+  which  is 
bound  by  it  at  the  same  moment,  otherwise  the  concentration  of 
silver  ions  in  its  saturated  solution  would  not  be  the  same  as  that 
of  the  N(Y  ions. 

All  of  these  conclusions  obviously  answer  to  the  formation  of 
the  electrically  neutral  compound: 

H 
I 
H3N.R.COAg.N.R.COO 

N03 
and  its  precipitation. 

The  last  of  the  above  conclusions  also  agrees  with  the  finding 
of  Bonamartini  and  Lombardi  (22),  that  in  neutral  solutions  egg- 
albumin  binds  the  entire  CuS04  molecule  to  form  an  insoluble 
compound,  although  in  alkaline  solutions  it  binds  an  excess  of 
copper,  forming  a  soluble  compound. 

Rohmann  and  Hirschstein  (105)  have  described  compounds 
of  casein  with  silver,  or  silver  nitrate,  formed  by  acting  upon 
solutions  of  casein,  neutral  to  phenolphthalein,  with  excess  of 
AgNOs.  The  resultant  precipitate  when  prepared  from  solutions 
of  sodium  caseinate  contained  85  X  10~5  equivalents  of  silver  per 
gram,  while  that  prepared. from  ammonium  caseinate  contained 
77  X  10-6  equivalents  of  silver.  It  will  be  recollected  (Cf.  Chap. 
V)  that  at  neutrality  to  phenolphthalein  ca*sein  neutralizes  80  X 

*  For  it  not  only  dissolves  in  water,  but  swells  in  water.     Cf.  Chap.  XII. 


HEAVY  METALS  141 

10~5  equivalents  of  base.  This  corresponds,  therefore,  with  the 
direct  interaction  of  one  molecule  of  silver  nitrate  with  one  molecule 
of  the  caseinate,  according  to  the  equation: 

H 
I 

H2N.R.CONa.N.R.COOH  +  AgNO3 
I 

OH  H 

I 

=  H2N.R.COAg.N.R.COOH  +  NaNO3. 
I 
OH 

This  compound  is  sparingly  soluble  in  water  (0.47  per  cent) 
and  its  solution  requires  the  addition  of  a  small  amount  of  NaOH 
before  it  becomes  neutral  to  phenolphthalein.*  If  to  this  solu- 
tion, neutralized  to  phenolphthalein,  more  AgN03  be  added,  a 
fresh  precipitate  is  obtained  containing  a  higher  percentage  of 
silver  than  the  first,  and  the  additional  silver  content  is  exactly 
equivalent  to  the  quantity  of  alkali  which  was  added  to  the  first 
to  render  it  neutral  to  phenolphthalein.  The  active  mass  of 
caseinate  in  this  reaction  is  directly  proportional  to  the  number  of 
—  COH.N—  groups  which  have  been  opened  up  by  combination 
with  a  base,  i.e.,  which  are  electrolytically  dissociated.  Conversely, 
undissociated  —COH.N—  groups  cannot  react  with  neutral  silver 
nitrate. 

The  compounds  which  proteins  form  with  zinc  sulphate  have 
been  studied  by  Lippich  (72)  who  considers  that  the  reaction 
between  zinc  sulphate  and  protein  results  in  an  equilibrium  which 
is  determined  by  a  variety  of  factors  amongst  which  he  enumerates 
ionization,  hydrolytic  dissociation,  complex  salt  formation,  etc. 
He  finds  that  serum  proteins  combine  with  zinc  in  alkaline  solu- 
tion to  form  insoluble  salts  which  dissolve  in  excess  (J  saturated) 
of  ammonium  sulphate.  This  soluble  salt  he  believes  to  be  of  the 
type  ZnS04-protein,  the  insoluble  salt  formed  at  a  higher  degree 
of  hydration  (absence  of  a  dehydrating  agent  such  as  ammonium 
sulphate)  being  of  the  type  Zn-protein. 

The  compounds  which   copper  salts  form   with  amino-acids, 

*  Since  AgOH  is  a  very  weak  base,  a  caseinate  of  silver  corresponding  to  the 
caseinate  of  a  strong  base  which  is  neutral  to  phenolphthalein,  would  neces- 
sarily be  acid  to  phenolphthalein. 


142  CHEMICAL  STATICS 

peptids,  peptones  and  proteins  have  been  extensively  investi- 
gated by  Kober  (57)  (58)  (59)  (60)  and,  more  recently,  by  Osborne 
and  Leaven  worth  (88).  Kober  has  shown  that  the  copper  salts 
of  amino-acids  in  alkaline  solutions  yield  their  copper  quanti- 
tatively in  the  form  of  a  precipitate  of  cupric  hydrate  on  heating 
the  solution  or  on  addition  of  an  excess  of  alkali.  Under  similar 
conditions  the  peptones  and  peptids  yield  little  or  no  precipitate. 
Osborne  and  Leaven  worth  have  shown  that  the  maximum  amount 
of  cupric  hydrate  which  edestin  or  gliadin  will  hold  in  solution 
exactly  corresponds  with  the  number  of  —  COHN—  groups  in 
the  molecule  of  these  proteins,  assuming  that  one  atom  of  copper 
combines  with  each  nitrogen  atom.  This  fact  would  appear 
to  be  at  variance  with  the  view  expressed  by  Kober  and  Sugiura 
(59)  that  the  pink  "biuret"  color  which  alkaline  solutions  of  the 
peptone  compounds  of  copper  yield  on  heating  is  due  to  a  union 
of  copper  with  four  nitrogen  atoms,  for  if  this  were  the  case  the 
development  of  the  pink  color  would  lead  to  the  precipitation  of 
an  excess  of  cupric  hydrate  previously  held  in  solution  by  the 
—  COHN—  groups  of  the  peptone. 

Neumann  (84)  has  studied  the  compounds  which  ovomucoid 
forms  with  various  metal  salts  (ZnCl2,  CuCl2,  MgCl2,  A1C13,  FeCl3) 
and  finds  that  in  alkaline  solutions  this  protein  forms  insoluble 
compounds  with  the  heavy  metal,  the  weight  of  the  metal  com- 
bined being  proportionate  to  the  molecular  weight  of  its  hydrox- 
ide. In  other  words,  ovomucoid  and  the  various  hydroxides  of 
the  heavy  metals  combine  in  stoichiometrical  proportions. 

Rona  and  Michaelis  (106)  have  shown  that  ferric  hydrate  will 
displace  Ca(OH)2  from  its  combination  with  casein,  forming 
insoluble  ferric  caseinate.  Benedicenti  and  Rebello-Alves  (15) 
(16)  have  shown  that  the  magnetic  properties  of  iron  in  solution 
or  even  of  finely  pulverized  iron  suspended  in  water  are  "masked" 
by  the  presence  of  proteins.  They  believe  that  in  the  latter 
case  this  phenomenon  is  attributable  to  a  direct  fixation  of  the 
metallic  ions  by  the  protein. 

Henze  (42)  has  described  an  interesting  chromogenic  protein 
in  the  blood  corpuscles  of  ascidians  in  which  vanadium  would 
appear  to  play  the  part  of  the  iron  in  the  haemoglobin  of  mam- 
malian corpuscles. 

2.  The  Compounds  with  the  Phosphoric  Acids.  —  It  was 
pointed  out  by  Graham  in  1861  that  metaphosphoric  acid  unites 


PHOSPHORIC  ACIDS  143 

with  gelatin  to  form  an  insoluble  compound  (34),  100  parts  of 
gelatin  uniting,  according  to  this  observer,  with  3.6  parts  of 
metaphosphoric  acid.  Graham  suggested  that  the  difference  be- 
tween the  behavior  of  metaphosphoric  and  other  phosphoric 
acids  in  this  respect  might  be  connected  with  the  possibly  col- 
loidal character  of  the  latter.*  This  suggestion  has  more  recently 
been  reiterated  by  Mylius  (82).  There  appears,  however,  no 
adequate  reason  for  adopting  such  a  conclusion.  Metaphosphoric 
acid  differs  from  orthophosphoric  acid  in  other  respects,  and  these 
differences  are  not  attributable  to  the  "colloidal"  character  of 
its  solutions.  As  Graham  pointed  out,  the  low  equivalent  of 
metaphosphoric  acid  which  combines  with  gelatin  suggests  that 
the  metaphosphoric  acid  enters  into  the  compound  in  a  crystal- 
loid and  not  a  colloidal  (polymerized)  form.  Colloidal  silicic  acid 
combines  with  gelatin  in  its  colloidal  form  and  the  compound  con- 
tains nearly  equal  weights  of  gelatin  and  of  silicic  acid  (Graham, 
loc.  cit.,  p.  206). 

The  compounds  of  metaphosphoric  acid  with  the  proteins 
were  at  one  time  regarded  with  peculiar  interest  on  account 
of  their  supposed  identity  with  the  nucleo-proteins  (69)  (74). 
Pohl  (96)  and  Kossel  (61),  however,  showed  that  these  compounds 
differ  fundamentally  from  the  true  nucleins  in  that,  on  hydrolysis, 
they  yield  no  purin  bases.  Thanks  to  the  work  of  Miescher, 
Kossel  and  their  pupils,  it  has  now  been  shown  that  the  nucleins 
are  not  protein  salts  of  phosphoric  or  of  metaphosphoric  acid 
but  of  a  complex  substituted  phosphoric  acid,  nucleic  acid  (6). 

The  compounds  of  the  protein  with  metaphosphoric  acid 
appear,  as  a  rule,  to  be  unstable  save  in  the  presence  of  excess 
of  the  acid  (Graham,  loc.  cit.,  Malfatti,  loc.  cit.) ;  if  this  excess  be 
removed  by  washing  then  the  compound  undergoes  decompo- 
sition, liberating  metaphosphoric  acid.  Progressive  removal  of 
the  free  acid  by  washing,  therefore,  results  finally  in  complete 
decomposition  of  the  product.  Conversely,  in  the  presence  of 
a  variable  excess  of  metaphosphoric  acid  the  quantity  which  is 
bound  by  a  protein  is  greater  the  greater  the  excess  (28). 

Bechhold  (14)  has  described  compounds  of  egg-albumin  with 

*  "It  will  be  an  interesting  inquiry  whether  metaphosphoric  acid  is  a 
colloid,  and  enters  into  the  compound  described  in  that  character,  or  is  a 
crystalloid,  as  the  small  proportion  and  low  equivalent  of  the  acid  would 
suggest,"  loc.  cit.  page  221. 


144  CHEMICAL  STATICS 

orthophosphoric    acid    prepared    by    treating    crystallized    egg- 
albumin,  dissolved  in  NaOH,  with  POC13. 

3.  Compounds  of  the  Proteins  with  Carbonic  Acid.  —  Siegfried 
(113)  has  described  a  special  form  of  combination  between  inor- 
ganic salts  and  amino-  or  polyamino-acids  which  is  probably 
destined  to  assume  considerable  importance  in  the  eyes  of  physi- 
ologists. He  observed  that  if  CO2  be  passed  through  solutions 
of  various  amino-acids  in  barium  hydrate,  provided  that  the 
total  concentration  of  Ba(OH)2  is  not  greater  than  twice  that 
of  the  amino-acid  no  precipitate  of  barium  carbonate  is  obtained. 
Similar  results  are  obtained  with  Ca(OH)2,  and  the  peptones 
and  the  proteins  of  the  blood  behave  in  a  manner  analogous  to 
the  simple  amino-acids.  On  standing,  BaC03  or  CaC03,  as  the 
case  may  be,  is  slowly  liberated,  and  this  process  is  accelerated 
by  heating.  Direct  analysis  of  the  products  obtained  when 
CaC03  acts  upon  glycocoll  and  other  amino-acids  showed  that  the 
compounds  can  be  represented  by  the  general  formula 

H 


R 

I  I 

COO  --  Ba 

being  the  barium  salts  of  carbamino-acids. 
For  the  monoamino-acids  the  ratio 

molecules  C02  bound 
atoms  of  N 

is  1,  indicating  that  the  —  NH2  group  reacts  quantitatively  with 
the  carbonate.  For  diamino-acids,  such  as  lysin,  the  ratio  is 
also  1,  showing  that  both  —  NH2  groups  react  quantitatively. 
For  arginin,  which  contains  4  atoms  of  nitrogen,  the  ratio  is  J, 
indicating  that  only  one  of  the  —  NH2  groups  and  neither  of  the 
imino-groups  react.  For  the  different  dipeptids  the  ratio  varies 
between 

1         ,     1 
L63  and  L79* 

If  the  —  N.HOC—  groups.  did  not  react  at  all  the  ratio  would  be 
i,  if  they  reacted  quantitatively  it  would  be  1.  For  tripeptids 

the  ratio  is  „-==,  whereas  it  would  be  £  if  the  —N.HOC—  groups 


ALKALOIDAL  REAGENTS,  ETC.  145 

did  not  react  at  all.    For  the  tetrapeptid  triglycyl  glycin  the 


ratio  is 


When  C02  is  passed  into  a  solution  of  a  protein  in  Ca(OH)2 
the  conductivity  of  the  mixture  diminishes.  From  this  Siegfried 
draws  the  conclusion  that  the  proteins  react  with  carbonates 
to  form  carbamino  acids.  From  what  has  been  said  in  the  pre- 
vious chapter  concerning  the  mode  of  combination  of  proteins 
with  salts,  however,  the  reader  will  gather  that  the  validity  of 
this  conclusion  does  not  admit  of  being  established  in  so  simple 
a  manner. 

4.  Compounds  of  the  Proteins  with  the  Alkaloidal  Reagents, 
Dyes,  Alkaloids,  etc.  —  In  order  that  a  protein  may  react  with 
an  acid  which  is  insufficiently  dissociated  to  break  up  its 
—  N.HOC—  groups,  some  stronger  acid  must  be  present,  com- 
bined with  the  protein,  and  which  can  then  be  displaced  by  the 
weaker  acid  through  the  agency  of  one  of  its  more  strongly  ionized 
salts.  Thus: 

H 

-N.HOC-  -h  HA  =  -N"+  HOC- 


H  H 

I          ++  I          ++ 

-N"  +  HOC-  +  BA'  =  -N"  +  HOC-  +  BA 
I  I 

A  A' 

HA  being  a  strong  acid,  HA'  a  very  weak  acid,  and  BA'  a  salt 
of  that  acid.  Similar  remarks  apply,  of  course,  to  very  weak 
bases.  Hence  it  is  observed  that  proteins  will  combine  with 
very  weak  acids  or  with  the  acid  radical  of  salts  more  readily 
in  acid  than  in  neutral  or  alkaline  solution,  and  with  very  weak 
bases  or  the  basic  radical  of  salts  in  an  alkaline  rather  than  a 
neutral  or  acid  medium.  Similarly  a  protein  may  not  be  able 
to  decompose  a  salt  of  an  acid,  binding  the  acid  (or  base  as  the 
case  may  be),  but,  in  the  presence  of  a  free  acid  it  may  be  able 
to  do  so,  partly  because  the  first  acid  is  partially  set  free  from 
its  salt  by  the  second,  but  also  because  the  ionization  of  the 
protein  is  increased. 


146  CHEMICAL  STATICS 

Hence,  on  adding  free  picric,  molybdic,  tungstic,  phospho- 
tungstic,  tannic,  stearic  or  chromic  acids  to  a  neutral  solution  of 
protein  a  precipitate  or  coagulum  is,  as  a  rule,  formed  immedi- 
ately. But  if  these  acids  be  added  in  the  form  of  their  (neutral) 
salts  then  no  precipitate  results  until  the  reaction  of  the  solution 
is  rendered  acid,  when  the  protein  at  once  combines  with  the 
acid  radical  of  the  salt.  Similarly,  neutral  lead  acetate  will 
not,  as  a  rule,  precipitate  protein  (egg-albumin)  from  neutral 
solutions,  but  it  will  precipitate  it  readily  from  faintly  alkaline 
solutions  (77)  (solutions,  that  is,,  in  which  the  protein  is  in  the 
form  of  an  ionized  compound  with  a  base). 

Upon  these  facts  probably  depend  certain  phenomena  which 
are  encountered  in  the  tanning  of  leather.  As  is  well  known, 
chromium  unites  firmly  with  the  hide-substance  only  in  the 
form  of  its  cation  Cr+++,  and  not  when  it  is  united  with  oxygen 
to  form  an  anion  as  in  chromic  acid.  Now  it  is  observed  that 
firm  union  only  occurs  when  the  chromium  salt  is  "basic"  (such 
as  Cr2Cl3(OH)3),  i.e.,  when  the  salt  is  neutral  in  reaction  or  even, 
at  the  moment  of  union,  faintly  alkaline  (97).  Further  addition 
of  alkali  results  in  a  displacement  of  the  chromium  by  the  added 
base.  On  the  other  hand,  tannic  acid  unites  with  the  hide  sub- 
stance in  a  faintly  acid  medium. 

Similar  principles  apply  to  the  union  of  dyes  with  proteins  (77) 
(39).  Proteins  which  are  about  equally  acid  and  basic  in  charac- 
ter, such  as  gelatin  and  egg-albumin,  combine  with  basic  dyes  only 
in  faintly  alkaline  solutions.  Predominantly  acid  proteins,  such 
as  casein  or  the  nucleins,  will  combine  with  basic  dyes  even  in 
neutral  or  faintly  acid  solutions  (80)  since  (ionized)  salts  of  these 
proteins  with  bases  can  exist  even  in  solutions  acid  to  litmus 
(Cf .  Chap.  V) ;  on  the  other  hand,  they  combine  with  acid  dyes 
with  great  difficulty.  Lilienfeld  has  shown  that  so  long  as  nucleic 
acid  is  not  saturated  with  albumins  to  form  neutral  nuclein  an 
insoluble  compound  with  methyl  green  is  formed  when  this  dye 
is  added  to  a  solution  of  the  nuclein,  but  after  saturation  with 
protein  the  nucleins  show  a  greater  affinity  for  acid  dyes  (71). 

An  interesting  compound  of  azolitmin  and  mucoid  has  been 
obtained  by  Rosenbloom  and  Gies  (109).  I  have  found  that  in 
faintly  alkaline  solution  trypsin,  or  some  constituent  of  Gruebler;s 
trypsin  (nach  Spateholz),  forms  an  insoluble  compound  with 
safranin  (99)  (44). 


ALKALOIDAL  REAGENTS,  ETC.  147 

Evidence  of  the  existence  of  protein  dye  compounds  even 
when  these  are  freely  soluble  is  afforded  by  the  influence  of  added 
protein  upon  the  distribution  of  the  dye  between  water  and  some 
other  appropriate  solvent  (100).  Thus  if  erythrosin  in  acidulated 
water  is  shaken  up  with  ethyl  acetate  it  passes  completely  into 
the  ethyl  acetate,  forming  a  yellow  solution  and  leaving  the 
water  colorless;  if,  however,  neutral  sodium  caseinate  be  added 
to  the  water,  the  watery  layer  remains  pink  upon  shaking  it  up 
with  erythrosin.  Similarly,  when  gentian  violet  in  alkaline 
watery  solution  is  shaken  up  with  ethyl  acetate  a  large  proportion 
of  the  gentian  violet  passes  into  the  ethyl  acetate,  forming  a 
red  solution  but  leaving  the  water  blue;  if,  however,  casein  or 
gelatin  be  added  to  the  water  very  little  of  the  gentian  violet 
passes  into  the  ethyl  acetate  and  that  which  does  so  only  stains 
it  faintly  violet.  Similarly  neutral  red  can  be  shown  to  combine 
with  casein,  and  bismarck  brown  with  casein  and  gelatin.  I 
have  observed  that  casein  forms  insoluble  compounds  with  acid 
fuchsin  and  orange  G,  gelatin  with  crystal  violet  and  acid  fuchsin, 
and  protamin  with  orange  G  and  carminic  acid.  The  compound 
of  protamin  with  carminic  acid  is  very  interesting  because  it  is 
of  a  very  different  color  from  the  free  acid  or  its  compounds 
with  inorganic  bases;  as  is  well  known,  free  carminic  acid,  in 
solution,  is  golden  in  color,  while  its  salts  with  inorganic  bases 
have  the  familiar  carmine  tint;  the  compound  with  protamin 
is,  however,  deep  violet.  I  have  observed  in  several  cases,  also, 
that  the  colors  of  solutions  of  dyes  to  which  the  proteins  have 
been  added  are  not  identical  with  the  colors  of  pure  watery 
solutions. 

Many  authors  have  doubted  the  chemical  nature  of  the  process 
of  staining  of  tissues,  preferring  to  regard  it  as  a  physical  phe- 
nomenon of  "solid  solution"  or  "adsorption."  Having  regard, 
however,  to  the  unquestionable  chemical  combinations  between 
dyes  and  proteins  which  occur,  as  we  have  seen,  in  vitro,  and 
recollecting  that,  as  Mathews  has  shown,  even  proteins  coagu- 
lated by  fixatives  show  similar  phenomena,  there  can  remain 
very  little  doubt  that  similar  combinations  must  frequently  occur 
between  the  dyes  used  by  cytologists  and  the  protein  constituents 
of  the  tissues.* 

*  For  a  discussion  of  this  interesting  question  Cf.  Gustav  Mann  (75),  also 
Pelet-Jolivet,  L.,  "Die  Theorie  des  Farbeprozesses,"  Dresden  (1910). 


148  CHEMICAL  STATICS 

That  casein  will  combine  with  various  alkaloids  to  form  water- 
soluble  compounds  has  been  shown  by  W.  A.  Osborne  (89). 
From  the  marked  effects  of  caffeine  upon  the  viscosity  and  con- 
ductivity of  gelatin  in  hydrochloric  acid  solutions  Pauli  and 
Falek  (93)  infer  that  caffeine  enters  into  combination  with  gelatin 
hydrochloride.  The  compounds  which  the  various  dyes  form 
with  amino-acids  have  been  studied  by  Suida  (116). 

5.  The  Compounds  of  Proteins  with  Soap  and  Lipoids.  —  It 
has  been  pointed  out  by  Rona  and  Michaelis   (106)  that  the 
power  of  soaps,  such  as  sodium  oleate,  to  reduce  the  surface 
tension  of  water,  is  much  diminished  by  the  presence  of  proteins, 
and  they  infer  that  the  soaps  form  compounds  with  protein. 
No  such  effect  was  observed  when  esters,  such  as  amyl  acetate, 
tributyrin,    propyl    acetate    or   ethyl   butyrate   were   employed 
instead  of  soaps.     These  results  have  been  confirmed  by  Berczeller 
(17).     It  has  long  been  known  that  it  is  an  extremely  difficult 
matter  to  free  proteins  from  contamination  with  lecithin,  and 
the  existence  of  lecithin-protein  compounds  has  frequently  been 
inferred  from  this  fact  (48)   (87)   (95).     More  decisive  evidence 
of  the  existence  of  such  compounds  has  recently  been  brought 
forward  by  Feinschmidt  (27)  and  Allemann  (5)  who  have  shown 
that  the  optimal  H+  concentration  for  the  precipitation  of  vari- 
ous proteins  is  very  considerably  modified  by  the  presence  of 
lecithin. 

Fatty  acid  compounds  of  the  peptones  have  been  prepared  by 
Izar  and  di  Zuattro  (54)  by  the  action  of  chlorides  of  the  fatty 
acids  upon  peptones  at  low  temperatures.  The  compounds  thus 
prepared  contain  from  18  to  20  per  cent  of  fatty  acid. 

6.  The  Compounds  of  Proteins  with  Proteins  and  their  Pos- 
sible Significance  in  Life  Phenomena.  —  It  has  been  shown  by 
Kossel   (62)   that  the  protamins,  which,  it  will  be  recollected, 
are  strongly  basic  proteins,  when  added  to  weakly  alkaline  solu- 
tions of  other  proteins  give  rise  to  precipitates.     Kossel  believes 
that  the  compounds  which  are  thus  formed  between  the  pro- 
tamins and  less  basic  proteins  are  analogous  to  the  naturally 
occurring  histones  (63)   (66).     Hunter  (53)  has  further  investi- 
gated these  compounds  -and  finds  that  while  crystallized  egg- 
albumin,  casein,   hemi-elastin,  gelatin,  edestin,  heteroalbumose, 
protalbumose,  "alkali-albuminate"  and  histone  sulphate  yield  a 
precipitate  in  alkaline  solution  upon  the  addition  of  clupein. 


COMPOUND  PROTEINS 


149 


Elastin,  peptone,  deuteroalbumose,  histopeptone  and  several 
peptids  fail  to  yield  a  precipitate.  Upon  digestion  of  these 
precipitates  with  pepsin  the  protamin  is  set  free  *  and  the  remain- 
der of  the  compound  is  converted  into  deuteroalbumose  and 
peptones  which  are  not  precipitable  by  picric  acid.  The  pro- 
tamin which  was  held  in  the  compound  before  digestion  can  now 
be  determined  quantitatively  by  precipitation  with  picric  acid. 
The  following  are  among  the  results  which  were  obtained  by 
Hunter : 


Protein  combined  with  clupein 

In  100  parts  of  nitrogen 
in  the  compound  the  pro- 
tamin moiety  contains 

Weight  of  protein  which 
combines  with  one  part 
by  weight  of  clupein 

Alkalialbuminate  

34.36 

Ovalbumin  
Gelatin  

29.31 
22.79 

4.1 

4.8 

Hemi-elastin 

22  71 

5  2 

Casein 

39  17 

2  5 

Edestin 

13  95 

8  5 

An  attempt  made  by  Gay  and  Robertson  (32)  to  prepare 
protamin  (salmin)  caseinate  by  the  method  of  Hunter  failed  to 
yield  a  product  containing  the  high  proportion  of  protamin  which 
he  describes.  The  nitrogen  content  of  the  compound  protein 
indicating  a  protamin  content  of  less  than  ten  per  cent.  The 
origin  of  this  discrepancy  has  been  ascertained  by  af  Ugglas 
(117)  who  has  shown  that  the  preparations  described  by  Hunter 
contained  a  considerable  excess  of  the  protamin  precipitated  in 
a  condition  of  admixture  with  the  compound.  The  compound 
of  protamin  (clupein)  with  casein  prepared  by  af  Ugglas  con- 
tained 94  per  cent  of  casein  and  6  per  cent  of  the  protamin 
while  the  compound  with  haemoglobin  contained  5  per  cent  of 
protamin.  Schmidt,  who  has  prepared  protamin  (salmin)  edesti- 
nate  (110)  (116),  finds  that  it  contains  about  ten  per  cent  of  the 
protamin. 

When  globin  and  casein  are  mixed  in  faintly  acid  solution 
(110)  a  precipitate  of  globin  caseinate  is  produced  which  is  soluble 
in  excess  of  acid  or  in  dilute  alkalies  (103).  That  this  precipi- 
tation is  accompanied  by  true  compound  formation  has  been 
demonstrated  by  the  method  of  electrometric  titration  (Schmidt 

*  The  protamins,  although  readily  digestible  by  trypsin,  are  not  digestible 
by  pepsin. 


150  CHEMICAL  STATICS 

(110)  (111)).  The  precipitate  produced  by  admixture  of  an 
excess  of  globin  with  sodium  caseinate  in  solution  contains  about 
34.5  per  cent  of  casein.  A  compound  of  globin  with  deutero- 
albumose  has  been  prepared  by  Schmidt  (111). 

Thymus  histone  combines  with  haemoglobin,  according  to 
af  Ugglas  (117),  in  the  proportion  of  one  part  of  thymus  histone 
to  two  of  haemoglobin,  and  with  casein  to  form  a  compound 
containing  about  30  per  cent  of  the  histone. 

A  particularly  interesting  compound  protein  is  the  haemo- 
globin caseinate  which  has  been  prepared  by  af  Ugglas.  To  a 
solution  of  casein  in  alkali  an  excess  of  hydrochloric  acid  is  added 
until  the  precipitate  of  free  casein  which  is  at  first  formed  is 
redissolved.  The  casein  hydrochloride  is  precipitated  from  this 
solution  by  the  addition  of  sodium  chloride  and  the  precipitate 
redissolved  and  reprecipitated  until  the  washings  from  the  pre- 
cipitate are  perfectly  neutral.  A  solution  of  this  substance 
added  to  an  excess  of  a  solution  of  haemoglobin,  produces  a  pre- 
cipitate containing  33  per  cent  of  casein  and  66  per  cent  of 
haemoglobin.  The  commonly  accepted  molecular  weight  of  haemo- 
globin, which  has  now  been  confirmed  by  a  variety  of  measure- 
ments (Cf.  section  11),  is  about  16,700.  We  have  seen  that  the 
minimal  molecular  weight  of  casein  is  about  8800  (Chap.  V). 
It  would,  therefore,  appear  that  casein  and  haemoglobin  combine 
with  one  another  in  molecular  proportions. 

From  important  observations  of  Hardy's  it  appears  extremely 
probable  that  many  of  the  protein  constituents  which  may  be 
isolated  from  the  various  tissues  and  tissue  fluids  do  not  pre- 
exist there  but  are  bound  up  in  complex  compounds  of  proteins 
with  proteins.  I  quote  from  the  appendix  to  Hardy's  article 
on  globulin  (35). 

"The  facts  of  the  case  are  these.  The  proteins  of  serum  are 
electrically  inactive.  Neither  the  whole  nor  any  fraction  moves 
in  a  field.  It  is  not  possible  to  detect  a  trace  of  'ionic'  protein. 
Dialysis  or  dilution  disturbs  the  equilibrium  and  'ionic'  globulin 
appears,  and  can  be  swept  out  of  the  general  mass  of  proteins  as 
an  opalescent  cloud  before  dialysis  has  been  pushed  to  the  point 
where  precipitation  occurs.  The  development  of  even  minute 
quantities  of  'ionic'  globulin  can  be  detected  in  this  way.  The 
direction  of  the  movement  is  towards  the  anode,  therefore  the 
globulin  which  appears  is  the  anion  of  alkali-globulin." 


COMPOUND  PROTEINS  151 

"Serum  which  has  been  dialyzed  against  water  with  very  low 
carbonic  acid  content  until  it  ceases  to  give  any  precipitate, 
but  which  can  still  give  with  acid  a  large  yield  of  globulin,  is 
in  a  most  interesting  condition.  The  whole  remaining  protein 
is  now  ionic.  It  moves  towards  the  anode  quite  uniformly, 
therefore  it  behaves  as  a  whole  as  the  protein  ion  of  an  alkali 
protein  compound." 

"Now  what  does  this  mean?  The  presence  of  other  proteins 
does  not  interfere  with  the  movement  of  ionic  protein.  This 
point  cannot  be  too  much  insisted  on.*  It  lies  at  the  root  of 
the  evidence.  Therefore  one  starts  with  protein  which  behaves 
uniformly  and  is  electrically  inactive,  one  ends  with  protein 
which  behaves  uniformly  and  is  electrically  active,  and  in  the 
final  stage  there  is  no  evidence  of  more  than  one  kind  of  protein 
ion.  But  this  residue  of  uniform  character  still  contains  a  globulin 
fraction.  It  can  be  split  by  saturation  with  a  neutral  salt  or 
by  acidulation  into  fractions  differing  in  properties  according  to 
the  mode  of  separation." 

"When  in  the  cell  used  for  these  experiments,  which  is  de- 
scribed on  p.  289,f  the  upper  layer  of  fluid  is  a  solution  of  ^^th 
normal  acetic  acid  and  the  lower  layer  is  serum  and  a  current 
is  passed  for  24  hours,  the  serum  protein  becomes  slowly  charged 
in  such  a  way  that  it  is  repelled  from  both  poles.  Therefore 
in  the  anodic  limb  it  becomes  charged  positively,  in  the  cathodic 
limb  negatively.  The  result  of  the  double  repulsion  is  that  the 
protein  is  condensed  into  a  hard  plate  of  rubber-like  consistency 
just  midway  between  the  electrodes." 

"The  phenomena  would  be  explained,  on  the  theory  which  has 
been  outlined,  in  this  way.  In  the  anodic  limb  an  excess  of 
hydroxyl  ions  are  liberated  owing  to  the  electric  convection,  and 
these,  reacting  with  the  serum  protein,  convert  it  into  cationic 
protein.  In  the  cathodic  limb  the  converse  reaction  with  hydrions 
occurs  and  is  possible  owing  to  the  amphoteric  nature  of  proteins. 
The  entire  mass  of  serum  protein  is  thus  thrown  into  the  ionic 
state  and  in  this  condition  moves  with  uniform  motion,  the  one 
half  as  cationic  protein,  the  other  half  as  anionic  protein.  The 
electric  current,  the  most  subtle  of  analyses,  detects  only  one 
substance,  and  this  substance,  owing  to  its  amphoteric  nature 
can  exist  in  either  the  cationic  or  the  anionic  state." 

*  Cf.  also  Chap.  XIII.    (Author.)        f  Cf.  Hardy's  paper.     (Author.) 


152  CHEMICAL  STATICS 

"The  concentrated  hard  rubber-like  mass  of  protein  obtained 
in  this  way  cannot  be  discriminated  from  serum  protein.  It 
is  easily  soluble  in  distilled  water  and  in  dilute  salt  solutions. 
From  the  solution  in  water  a  globulin  fraction  can  be  precipitated 
by  acid,  or  by  saturation  with  magnesium  sulphate.  From  the 
solution  in  dilute  salt  solution  a  globulin  fraction  can  be  pre- 
cipitated by  saturation  with  salt  (NaCl,  or  MgSO^.  The  ni- 
trate, after  removal  of  the  globulin  by  saturation,  contains  a 
protein  which  is  precipitated  by  acetic  acid." 

"The  position  is  that  in  serum  one  has  protein  which  can  be 
thrown  into  the  ionic  state  and  which  then  moves  in  a  field  as  a 
single  substance.  From  it  an  electrically  active  fraction,  namely 
globulin,  can  be  split  off,  and  the  protein  thereby  becomes  elec- 
trically heterogeneous." 

"Now  the  globulin  fraction  has  an  abiding  characteristic.  In 
all  its  solutions  its  molecular  state  is  so  gross  as  to  cause  the 
molecules  to  be  arrested  by  a  porous  pot.  They  will  not  pass 
such  a  filter  even  under  pressure.  In  this  it  is  sharply  distinct 
from  the  parent  serum  protein,  which  is  readily  filterable.  If 
globulin  be  present  as  such  in  serum  it  is  not  only  non-ionic,  but 
the  agent  which  dissolves  it  must  be  something  more  than  alkali 
and  salt  since  either  alone  or  together  they  will  not  produce  so 
high  a  grade  of  solution  (78)." 

"The  difference  in  the  molecular  grade  of  globulin  when  once 
separated,  and  the  electrical  homogeneity  of  serum  protein  and 
of  the  fraction  (still  capable  of  further  subdivision  by  salting 
out)  which  remains  after  the  alkaline  globulin  fraction  which 
most  readily  appears  has  been  removed  suggests  that  serum 
protein  is  a  complex  unit.  If  such  a  unit  exists  it  is  not  satu- 
rated with  globulin.  Fresh  ox-serum  has  an  extraordinary  power 
of  dissolving  globulin,  it  will  take  up  almost  its  own  volume  of 
the  thick  cake  at  the  bottom  of  a  centrifuge  tube;  and  in  ox- 
serum  so  saturated  there  is  not  a  trace  of  alkali  globulin  nor  of 
any  ionic  protein." 

The  phenomena  observed  by  Hardy  appear  to  admit  of  in- 
terpretation on  the  view  (102)  that  the  protein-complex  in  serum 
is  formed  by  the  union  of  a  number  of  alkali  protein  compounds, 
the  union  taking  place  in  a  manner  strictly  analogous  to  the 
combination  of  neutral  salts  with  protein  (Cf.  Chap.  VI),  alkali 
protein  molecules  behaving  like  inorganic  salt  molecules  thus: 


COMPOUND  PROTEINS  153 

H  H 

I  I 

2HOOC.Ri.N"  +  2-H-NaOC.Ri.NH2  +  HOOC.R.N"  + 
I  I 

OH  OH 

RLCOOH 

I 

H     N  =  NaOC.R!.NH2 

++NaOC.R.NH2  =  HOOC.R.N  (     )  NaOC.R.NH2     +  2  H2O 

I   \/ 

OH  N  =  NaOCRi.NH2 
I 
Rx.COOH 

Hydrolytic  dissociation  of  this  complex,  just  as  in  the  case  of  the 
salt-alkali-protein  compounds  described  in  the  previous  chapter, 
would  result  in  its  decomposition,  partial  or  complete,  and  hence, 
if  the  complex  were  non-ionic  (and,  as  we  have  seen,  the  homol- 
ogous compounds  with  inorganic  salts  are  non-ionic)  mere  dilu- 
tion of  its  solution  or  the  removal  of  dehydrating  agents  (salts) 
might  result,  as  in  Hardy's  experiments,  in  the  splitting  off  of 
fraction  after  fraction  of  ionic  protein.  A  very  faintly  alkaline 
reaction  would  probably  favor  its  stability,  a  mere  trace  of  acid 
might  be  expected  to  disrupt  the  complex  (Cf.  equation  iv,  Chap. 
VI).*  It  will  be  found,  I  believe,  that  the  presence  of  such 
protein-complexes  as  these,  in  the  tissues  and  tissue-fluids,  affords 

*  The  fact  that  this  complex  will  pass  through  the  pores  of  a  porcelain 
filter,  while  the  simpler,  ionic  protein  will  not,  is  due  to  its  non-ionic  character. 
The  thesis  will  be  developed  in  a  later  chapter  (Chap.  XI),  upon  a  very 
extensive  experimental  basis,  that  the  colloidal,  non-filterable,  viscous  character 
of  solutions  of  ionic  protein  is  attributable  not  to  the  size  of  the  protein  particles 
but  to  the  size  of  the  associated  complex  of  water  molecules,  which  is  much  larger 
in  solutions  of  ionic  than  in  solutions  of  non-ionic  protein.  The  non-filterable 
character  of  ionic  globulin  is  to  be  interpreted  in  the  same  way  as  the  non- 
permeability  of  a  porous  pot  for  mixtures  of  propyl  alcohol  and  water,  although 
it  is  permeable  to  both  water  and  propyl  alcohol  when  they  are  pure  (S.  U. 
Pickering  (94)).  Just  as,  in  Pickering's  experiment,  propyl  alcohol,  when  dis- 
solved in  water,  will  not  pass  out  through  the  walls  of  a  porous  pot  into  the 
surrounding  water,  not  because  the  wall  of  the  pot  is  impermeable  to  propyl 
alcohol,  for  pure  propyl  alcohol  will  pass  through  the  pot,  but  because  it  is 
impermeable  to  the  molecule  of  propyl  alcohol  plus  its  associated  complex  of 
water  molecules,  so  ionic  globulin  will  not  pass  through  porcelain  because  the 
porcelain  is  impermeable  to  the  globulin  ion  plus  its  associated  complex  of 
water  molecules. 


154  CHEMICAL  STATICS 

a  simple  and  readily  intelligible  explanation  of  an  otherwise 
exceedingly  puzzling  fact.  I  refer  to  the  individuality  of  the 
tissues  and  tissue-fluids.  Despite  the  fact  that  the  individual 
proteins  which  are  found  in  the  tissue-fluids  of  tolerably  nearly 
related  animals  (e.g.,  the  mammalia)  appear,  on  analysis,  to  be 
identical  with  one  another,*  yet  the  tissue-fluids  of  one  mammal, 
when  injected  into  the  circulation  of  another,  are  treated  as 
foreign  intrusions  and  give  rise  to  "antibodies";  while  the  indi- 
vidual protein  constituents  of  these  fluids  are  also  treated  as 
foreign  matter  when  introduced  into  the  circulation,  giving  rise 
to  "  precipitations "  and  to  phenomena  such  as  "  anaphylaxis " 
(120). 

On  the  basis  of  the  view  developed  above,  however,  not  only 
the  constitution  of  the  individual  components  of  the  protein 
complex  is  of  importance  in  determining  its  characteristics,  but 
also  the  relative  proportion  of  these  components.!  Two  protein- 
complexes  of  this  type  might  well  be  built  up  out  of  identical 
units  and  yet  differ  fundamentally  owing  to  differences  in  the 
combining  proportions  and,  consequently,  in  the  mode  of  linkage 
of  these  units.  Any  one  constituent  of  the  complex  would,  of 
course,  be  a  totally  different  substance  from  the  complex  itself 
and  its  introduction  into  the  tissues  or  tissue-fluids  would  result 
in  a  more  or  less  extensive  disruption  of  the  equilibrium  of  which 
the  complex  is  an  expression  and  which  lends  it,  in  each  tissue, 
tissue  fluid  and  species,  its  own  distinctive  character. 

In  pursuance  of  this  idea  Gay  and  Robertson  (32)  (33)  and 
C.  L.  A.  Schmidt  (110)  (111)  have  investigated  the  antigenic 
properties  of  several  compound  proteins.  If  compound  proteins 
differ  in  their  biological  specificity  from  their  constituents  then 

*  Thus  the  casein  of  human  milk  is  chemically  identical  with  the  casein  of 
cows'  milk  and  with  that  of  goats'  milk  (2)  (1).  The  fibrin  of  ox-blood  ap- 
pears to  be  identical  with  that  of  horse-blood  (4).  The  serum  albumins  and 
globulins  of  goose-blood  are  identical  with  those  of  horse-blood  (3).  Whether 
chemically  identical  proteins  derived  from  different  animal  species  are  also 
antigenically  identical  or  not  has  not  yet  been  satisfactorily  established. 

t  Just  as  the  different  proteins  and  polypeptids  are  built  up  out  of  very 
similar  amino-acids  and  yet  differ  widely  from  one  another  in  their  character- 
istics because  the  proportions  in  which  the  amino-acids  enter  into  the  mole- 
cules are  different.  These  protein-complexes  may  be  regarded  as  bearing  the 
same  relationship  to  the  simple  proteins  which  they  yield  on  decomposition  as 
the  polypeptids  do  to  the  amino-acids. 


COMPOUND  PROTEINS  155 

a  compound  protein  should  represent  a  new  antigen  giving  rise 
to  antibodies  for  itself  as  distinguished  from  the  antibodies  for 
its  constituents.  Unfortunately  a  formidable  technical  difficulty 
stands  in  the  way  of  clearly  recognizing  the  presence  of  anti- 
bodies which  are  specific  for  the  compound  protein.  This  is  the 
difficulty  which  is  constituted  by  the  fact  that  any  protein  which 
is  capable  of  being  split  by  hydrolysis  into  moieties  which  are 
still  proteins  (in  the  sense  that  they  are  antigenic)  gives  rise  on 
injection  into  animals  to  antibodies^  not  only  for  itself,  but  also 
for  these  split-products  (31).  Analogously,  a  compound  protein 
gives  rise  to  antibodies  for  its  constituent  parts  and  it  is  only 
possible  to  distinguish  between  these,  which  would  appear  in 
the  blood  of  immunized  animals  after  injection  of  the  separate 
constituents,  and  any  antibodies  which  may  be  formed  for  the 
compound  as  a  whole  in  those  doubtless  exceptional  instances 
in  which  the  antibody  for  the  compound  reacts  with  a  constituent 
which  is  not  normally  antigenic. 

We  have  therefore  investigated  from  this  point  of  view  certain 
compound  proteins  in  which  one  constituent  is  non-antigenic, 
such  as  protamin  caseinate,  of  which  the  protamin  constituent 
is  non-antigenic  and  toxic  while  the  casein  constituent  is  anti- 
genic and  non-toxic,  and  globin  caseinate  of  which  the  globin 
constituent  is  toxic  and  non-antigenic.* 

Protamin  caseinate  displays  no  antigenic  characteristics  which 
enable  it  to  be  distinguished  from  casein.  It  is  non-toxic,  but 
whether  this  lack  of  toxicity  is  attributable  to  the  masking  of 
the  toxic  properties  of  protamin  by  its  combination  with  casein, 
or  to  the  smallness  of  the  proportion  of  protamin  contained  in 
the  compound  has  not  yet  been  definitely  established.  It  gives 
rise  to  antibodies  for  casein  by  virtue  of  its  casein-content,  just  as 
casein  gives  rise  to  antibodies  for  its  split-product  paranuclein,  but 
it  does  not  give  rise  to  antibodies  for  protamin.  Similarly,  pro- 
tamin edestinate  (111)  is  non-toxic  and  gives  rise  to  antibodies 
for  edestin  but  does  not  give  rise  to  any  antibody  which  will 
react  with  its  protamin  constituent.  Globin  caseinate,  however, 

*  It  is  asserted  by  C.  H.  Browning  and  G.  H.  Wilson  (23)  that  globin  is 
antigenic.  The  negative  results  of  Gay  and  Robertson  which  have  been 
repeated  and  confirmed  by  Schmidt  show  that  the  antigenic  character  of  the 
globin  prepared  by  Browning  and  Wilson  must  have  been  due  to  contamination 
by  protein  impurities. 


156  CHEMICAL  STATICS 

differs  very  markedly  in  its  antigenic  behavior  from  either  of 
its  constituents.  In  the  first  place  it  is  non-toxic,  and  the  failure 
to  exhibit  toxicity  can  hardly  be  attributable  to  dilution  of  the 
globin  constituent  by  admixture  with  casein  since,  as  we  have 
seen,  globin  caseinate  contains  65.5  per  cent  of  globin  (103). 
Still  more  striking  is  the  fact  that  it  yields  antibodies  which 
react  (i.e.,  display  alexin-fixation)  not  only  with  the  casein  con- 
stituent of  the  compound,  but  also  with  the  globin  constituent. 
It  would  appear  evident,  therefore,'  that  injection  of  globin 
caseinate  into  animals  gives  rise  to  an  antibody  which  does  not 
appear  in  response  to  separate  injections  of  its  constituents. 
Schmidt  (111)  has  investigated  the  antigenic  behavior  of  a  com- 
pound of  globin  (toxic  and  non-antigenic)  with  deutero-albumose 
(non-toxic  and  non-antigenic) .  The  compound  retains  the  toxicity 
of  globin  and  is  non-antigenic. 

7.  Compounds    of    the    Proteins    with    Toxins,    Antibodies, 
Ferments,  etc.  —  Very  extensive  evidence  has  been  advanced  by 
numerous  authors  *  in  support  of  the  view  that  the  antitoxins 
are  true   proteins.     If  this  be  true,   and  the  probabilities  are 
immensely  in  favor  of  its  truth,  then  the  entire  series  of  toxin- 
antitoxin  reactions  are  reactions  in  which  proteins  play  a  leading 
part.     To  consider  these  here  would  be  manifestly  out  of  place; 
for  an  analysis  of  the  physico-chemical  laws  which  govern  these 
reactions  the  reader  is  referred  to  Arrhenius  (8). 

The  question  of  the  occurrence  of  combinations  between  the 
proteolytic  ferments  and  proteins  and  their  significance  in  the 
mechanism  of  protein  hydrolysis  will  fall  under  consideration 
in  a  later  chapter  (Chap.  XVI)  but  the  reader's  attention  is  here 
drawn  to  the  work  of  Hedin  (37)  (38)  upon  this  subject. 

8.  Methyl  and  Benzoyl  Derivatives  of  the  Proteins.  —  Ac- 
cording to  Rogozinski  (104),  methylation  of  clupein  causes  pro- 
found alteration  in  the  composition  of  the  molecule,  reducing 
especially  the  proportion  of  arginin  yielded  on  subsequent  hy- 
drolysis.    Skraup  and  Krause  (114)  have  shown  that  not  only 
is  arginin  reduced  but  also  tyrosin,  lysin  and  histidin,  while  the 
glutamic  acid  and  leucin  yields  are  unaffected. 

Blum  and  Umbach  (19). have  prepared  benzoyl  derivatives  of 
native  and  iodized  proteins. 

"  Cf.  for  literature  Carl  Oppenheimer  (86). 


HALOGEN  AND  NITRO  COMPOUNDS  157 

9.  The  Halogen  and  Nitro  Substitution-Compounds  of  the 
Proteins.  —  Besides  the  combinations  of  the  proteins  with 
inorganic  substances  which  are  formed  through  the  ionization 
of  —  N.HOC—  groups,  other  types  of  combination  between 
proteins  and  inorganic  substances  are  unquestionably  possible. 
Among  these  the  best  known  are  the  substitution-compounds  of 
the  proteins  with  the  halogens  and  with  N(>2. 

According  to  Blum  (18)  (20)  and  to  Hofmeister  (43)  (112)  (41) 
(85),  the  halogen  compounds  are  formed  by  the  replacement 
of  hydrogen  atoms  in  the  aromatic  groups  of  the  proteins  by 
halogen  atoms.  The  best  known  examples,  both  artificially  pre- 
pared and  naturally  occurring,  are  the  iodoalbumins.  They  can 
be  prepared  (Cf.  papers  just  cited)  by  allowing  a  mixture  of 
potassium  iodate  and  potassium  iodide  to  act  upon  proteins  at 
a  moderately  high  temperature  (40  degrees).  Hopkins  and 
Brook  (46),  however,  prepared  iodoproteins  by  allowing  powdered 
iodine  to  act  directly  on  the  protein,  in  solution.  When  the 
former  method  of  preparation  is  employed,  part  of  the  iodine 
enters  into  combination  with  the  protein  and  part  is  liberated 
as  HI.  Hence  excess  of  a  carbonate,  of  an  alkali,  or  of  an  alka- 
line earth  must  be  added  to  the  mixture  to  neutralize  the  hydro- 
gen iodide  set  free,  and  prevent  it  from  hydrolysing  the  protein, 
through  the  catalytic  action  of  hydrogen  ions.  The  iodo-proteins 
are,  in  the  dry  condition,  similar  in  appearance  to  ordinary  pro- 
teins; they  are  insoluble  in  water  or  alcohol,  and  they  appear 
to  be  more  predominantly  acid  than  the  non-iodized  proteins 
from  which  they  are  derived,  since  they  are  readily  soluble  in 
dilute  solutions  of  alkalies  or  of  alkaline  carbonates,  but  are 
soluble  in  acid  only  in  the  presence  of  a  considerable  excess. 

The  percentage  of  iodine  contained  in  the  iodo-proteins  varies, 
as  might  be  expected,  with  the  nature  of  the  protein,  —  it  also 
appears  to  vary  somewhat  with  the  mode  of  preparation  or 
subsequent  treatment,  since  two  types  of  compounds  are  formed, 
the  one  containing  a  very  high  percentage  of  iodine,  partly  in 
firm  and  partly  in  loose  combination  (the  per-iodo-casein  of 
Liebrecht  (70)),  and  the  other  a  lower  percentage  of  iodine  en- 
tirely in  firm  combination. 

Improved  methods  of  preparing  iodized  proteins  have  recently 
been  described  by  Oswald  (92). 

The  bromine  and  chlorine  substitution-products  of  the  proteins 


158  CHEMICAL  STATICS 

have  been  specially  studied  by  Hopkins  (45)  (46)  (47)  and  Blum 
and  Vaubel  (20).  The  following  were  the  percentages  of  the 
different  halogens  found  by  Hopkins  and  by  Blum  in  firm  com- 
bination with  egg-albumin.* 


Hopkins 

Blum 

6.2  per  cent  iodine. 
2.84  per  cent  bromine. 
1.93  per  cent  chlorine. 

6-7  per  cent  iodine. 
4-5  per  cent  bromine. 
2     per  cent  chlorine. 
1.2  per  cent  fluorine. 

As  in  the  case  of  the  iodo-proteins,  two  series  of  bromine  com- 
pounds exist:  the  one  in  which  the  bromine  is  firmly  bound  and 
the  percentage  of  bromine  is  low,  the  other  in  which  a  part  of 
the  bromine  is  loosely  bound  and  the  percentage  is  high.  Hop- 
kins and  Brook  found  in  this  latter  type  of  brominated  egg- 
albumin  14.89  per  cent  of  bromine.  These  compounds,  as  also 
the  chlorinated  egg-albumin,  are  soluble  in  hot  absolute  alcohol, 
the  halogen-protein  being  precipitated  from  this  solution  by  the 
addition  of  ether  (Hopkins  and  Brook). 

The  best-known  naturally  occurring  halogen-protein  is  the 
thyreoglobulin  of  the  thyroid  gland.  It  contains  only  1.75  per 
cent  of  iodine  (91).  The  physiological  action  of  this  protein 
does  not  appear  to  be  primarily  dependent  upon  its  iodine  con- 
tent (108)  and  it  is  doubtful  whether  this  is  the  physiologically 
active  iodine-containing  principle  of  the  thyroid  (56) .  An  iodized 
keratin,  gorgonin,  is  found  in  corals  (26)  (40)  (79)  (81).  It 
contains  a  high  (about  8)  percentage  of  iodine.  Sponges  also 
contain  iodo-proteins  (36)  (52)  (115). 

Nitro  substitution-products  of  the  proteins  were  first  prepared 
by  Loew  (73).  VonFtirth  (29)  prepared  them  by  acting  upon 
the  protein  with  nitrous  acid,  at  the  same  time  adding  urea  to 
prevent  the  formation  of  nitric  acid.  He  obtained  a  product  con- 
taining 1.78  per  cent  of  N02.  The  nitro  derivatives  of  the  pro- 
tamins  have  been  very  extensively  studied  by  Kossel  (65)  (64) 
(67)  (68)  (119),  who  believes  that  the  nitration  of  protein  leads 
to  the  entry  of  N02  into  the  guanidine  radical  of  the  arginin. 
The  nitrated  proteins  therefore  yield  nitroarginin  on  hydrolysis. 
On  treatment  with  alkali  the  nitro-guanidine  radical  breaks  up 
yielding  carbon  dioxide,  nitrous  oxide  and  ammonia. 

*  Cited  after  Gustav  Mann  (76). 


SULPHUR  159 

10.  The  Compounds  of  Proteins  with  Sulphur.  —  Uhl  (118) 
has  prepared  sulphur  compounds  of  the  proteins  by  utilizing 
the  reaction: 

<H 

I  XR  +  CS2=  I      R 

H  C  =  S 

I 
SH 

which  occurs  in  alkaline  solution.  These  compounds  combine 
with  heavy  metals;  the  salts  thus  formed  contain  a  high  pro- 
portion of  the  heavy  metal  and  are  soluble  in  water. 

11.  The  Compounds  of  Proteins  with  Oxygen.  —  The  best 
known  compound  of  this  type  is  that  of  oxygen  with  the  coloring 
matter   of   the   blood,   haemoglobin.     This   has   been   especially 
studied  by  Hiifner  (50)  who  employed  the  spectrophotometric 
method  of  measurement,  and  found  that  at  high  pressures  (by 
interpolation)  the  amount  of  oxygen  bound  up  by  one  gramme  of 
haemoglobin  is  1.338  cc.,  reduced  to  standard  temperature  and 
pressure.     Carbon    monoxide    combines    with    haemoglobin    in 
equivalent   proportions.     Hiifner   considered   that   the   reaction 
between  haemoglobin  and  oxygen  is  a  balanced  reaction,  namely, 
Hb  -j-  02  ?=*  Hb02.     From  this,  if  the  concentration  of  reduced 
haemoglobin  be  Cr  and  of  oxyhaemoglobin  C0  and  that  of  oxygen 


where  P0  is  the  partial  pressure  of  oxygen  and  at  the  absorption- 
coefficient  of  the  gas  at  temperature  t  it  would  follow  that  if  K 
is  the  velocity  constant 

Co       =Kat 
CrXP0      760' 

the  quantities  on  the  right  of  this  equation  being  constant  at 
constant  temperatures,  the  relation  between  the  oxygen  tension 
and  the  percentage  of  haemoglobin  converted  into  oxyhaemoglobin 
should  be  expressed  by  a  rectangular  hyperbola.  Bohr  (21), 
however,  found  that  the  curve  of  dissociation  of  oxyhaemoglobin 
is  not  a  rectangular  hyperbola,  and  he  explains  his  results  by 
supposing  that  the  haemoglobin,  in  the  presence  of  oxygen,  first 
splits  off  a  haematin-containing  moiety  and  that  this  then  sub- 


160  CHEMICAL  STATICS 

sequently  unites  with  the  oxygen.  Barcroft  and  Roberts  (9)  (10) 
(11)  (12)  (13)  have,  however,  shown  that  the  irregularities  ob- 
tained by  Bohr  were  due  to  the  presence  of  electrolytes  in  the 
haemoglobin  solutions;  in  dialysed  solutions  the  curve  of  dis- 
sociation is  exactly  that  demanded  by  Hiifner's  theory.  The 
velocity  of  dissociation  of  oxyhsemoglobin  obeys  the  equation 
indicated  by  the  mass  law.  The  variations  of  the  equilibrium- 
constant  (K)  with  change  of  absolute  temperature  follow  the 
van't  Hoff  equation 


K    dt       2T2 

where  q  is  constant  and  equal  to  28,000  calories.  This  is  there- 
fore the  heat  of  combination  of  one  gram-molecule  of  haemoglobin 
with  oxygen.  Since  the  amount  of  heat  which  is  actually  given 
out  when  one  gram  of  haemoglobin  unites  with  oxygen  is  1.85 
calories,  the  molecular  weight  of  haemoglobin,  in  dialysed  solu- 
tion is,  according  to  these  results,  15,000.  The  minimum  weight 
indicated  by  Hiifner's  results,  cited  above,  is  16,669.  A  similar 
figure  is  indicated  by  the  iron-content  of  the  haemoglobin  mole- 
cule (55)  (121)  (49)  (50)  and  by  the  osmotic  pressure  measure- 
ments of  Hufner  and  Gausser  (51).  Weymouth  Reid  (98), 
however,  found  a  value,  as  indicated  by  osmotic  pressure  meas- 
urements, three  times  as  great.  Barcroft  and  Hill  (11)  suggest 
that  haemoglobin  may  possibly  exist  as  a  polymer  of  itself,  under 
certain  conditions,  and  that  the  irregularities  observed  in  the 
curve  of  dissociation  of  oxyhaemoglobin  in  the  presence  of  elec- 
trolytes may  be  due  to  the  breaking  down  of  such  aggregates. 
The  temperature-coefficient  of  the  dissociation  of  oxyhaemoglobin 
is  large,  about  4  per  10  degrees  rise  in  temperature. 

These  masterly  investigations  of  Barcroft  effectually  demon- 
strate two  things;  in  the  first  place  that  the  union  between 
haemoglobin  and  oxygen  is  not  an  "adsorption  combination" 
as  Wo.  Ostwald  has  suggested  (90)  (101)  (.13)  and,  in  the  second 
place  that,  since  the  derivation  of  the  van't  Hoff  law  (reaction 
isochore)  involves  the  assumption  that  the  gas  laws  apply  strictly 
to  the  system  under  consideration,*  haemoglobin,  although  a 
colloid,  does  not  form  a  'separate  phase  within  its  solution,!  but 

*  Cf.  W.  Nernst  (83). 

t  It  may  be  remarked  that  under  such  conditions  haemoglobin  cannot 
present  any  surface  with  which  to  "adsorb." 


OXYGEN  161 

is  molecularly  distributed  throughout  the  solution  in  accordance 
with  the  law  of  Avogadro.  Again  we  see,  therefore,  as  we  saw 
in  considering  the  copper  albuminates  (Chap.  VI,  section  5),  that 
the  distinction  which  has  been  built  up  by  certain  authors  be- 
tween the  crystalloids  which  form  homogeneous,  and  the  colloids 
which  are  supposed  to  form  heterogeneous,  solutions,  is  entirely 
artificial  and  not  in  accordance  with  the  facts. 

The  results  obtained  by  Barcroft  and  his  co-workers  have 
received  decisive  confirmation  at  the  hands  of  Butterfield  (24) 
who,  using  the  spectrophotometric  method,  has  shown  with  a 
very  high  degree  of  precision,  that  the  absorption-spectrum  of  a 
solution  containing  a  mixture  of  oxyhsemoglobin  and  reduced 
haemoglobin  does  not  alter  upon  dilution.  The  solubility  of 
oxygen  in  water  not  being  appreciably  affected  by  the  presence 
of  haemoglobin  the  relative  proportion  of  the  reacting  substances 
(oxygen,  haemoglobin  and  oxyhsemoglobin)  is  not  altered  by 
dilution  and  therefore  in  accordance  with  the  mass  law  the  pro- 
portion of  oxygen  in  combination  remains  likewise  unaffected. 

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PART  II 
THE  ELECTROCHEMISTRY  OF  THE  PROTEINS 


CHAPTER  VIII 
THE   FORMATION   AND    DISSOCIATION   OF  PROTEIN   SALTS 

1.  Compounds  of  the  Proteins  with  Inorganic  Bases  and 
Acids ;  the  Non-Dissociable  Character  of  the  Inorganic  Radical. 
— -We  have  already  had  occasion  to  incidentally  dwell  upon  the 
fact  that  the  inorganic  radical  of  the  protein  base  and  protein 
acid  compounds  is  not  electrically  dissociated  as  such.  The  most 
direct  proof  which  we  possess  of  this  fact  is  that  which  was  ob- 
tained by  Bugarszky  and  Liebermann  (4).  These  observers 
employed  the  potentiometric  method,  using  two  different  con- 
centration chains.  The  first  was  the  ordinary  gas-chain: 


Pt  saturated  with  Hz 

Acid  (HC1) 

Base  (NaOH) 

Pt  saturated  with  H2 

1 

2 

3 

4 

the  potential  being  measured,  first  with  pure  acid  in  2  and  then 
with  acid  plus  protein;  the  difference  between  the  two  readings  * 
yielding  by  computation  from  the  Nernst  formula,  the  number 
of  hydrogen  ions  bound  by  the  protein.  The  second  concentration- 
chain  was  built  up  as  follows: 


Hg 

HgCl  (solid),  HC1 

NaCl 

NaBr,  HgBr  (solid) 

Hg 

1 

2 

3 

4 

5 

and  enabled  them  to  estimate,  in  a  similar  way,  the  number  of  Clf 
ions  bound  by  the  protein.  Now  the  number  of  CV  ions  bound  by 
a  given  mass  of  protein  dissolved  in  dilute  HCl  was  found  to  be 
exactly  equal  to  the  number  of  H+  ions  which  it  binds.  The  fol- 
lowing data  are  compiled  from  those  obtained  by  Bugarszky  and 
Liebermann : 

*  Less  a  correction  expressing  the  potential  between  2  and  3. 

167 


168 


ELECTROCHEMISTRY 


EGG-ALBUMIN  IN  0.05  N  HC1. 


Grams  protein  in  100 
cc. 

Per  cent  of  H+  bound 
by  the  protein 

Per  cent  of  Cl'  bound 
by  the  protein 

0 

0 

0 

0.4 

9.0 

10.7 

0.8 

18.9 

20.2 

1.6 

33.3 

38.0 

3.2 

60.2 

64.0 

6.4 

96.6 

76.0 

The  slight  irregularities  are,  save  in  the  last  observation,  no 
greater  than  might  have  arisen  out  of  the  uncertain  magnitude 
of  the  correction  for  the  potential  between  the  elements  2  and  3 
in  the  gas-chain. 

These  striking  results  have  been  confirmed  by  Rohonyi  (31) 
employing  Merck's  crystallized  ovalbumin  and  Witte's  peptone; 
the  following  were  the  results  obtained,  the  substances  being 
dissolved  in  JV/20  HC1  solution : 


Substance 

Per  cent 

Per  cent  of  chlorine 
ions  bound 

Per  cent  of  hydrogen 
ions  bound 

Ovalbumin  
Albumose  
Alanin 

1.9 
1.8 
1  5 

21.9 
37.2 
37  9 

22.6 
35.9 
66  2 

A  decided  difference  between  the  modes  of  combination  of  pro- 
teins and  an  amino-acid  with  hydrochloric  acid  is  thus  very 
clearly  revealed. 

Manabe  and  Matula  (17)  and  Blasel  and  Matula  (2)  have 
shown  that  at  low  H+  ion  concentrations  a  greater  proportion 
of  H+  is  bound  by  serum  albumin  or  gelatin  than  of  Cl'.  Ringer 
(33)  has  confirmed  this  observation  for  albumoses,  but  he  also 
finds  that  in  higher  concentrations  of  hydrochloric  acid  (1  per 
cent  of  albumose  in  0.1  N  HC1)  the  H+  and  Cl'  are  bound  equally. 
The  same  tendency  is  shown  in  the  observations  of  Bugarszky 
and  Liebermann  which  are  quoted  above,  for  in  the  solution 
containing  the  lowest  proportion  of  hydrochloric  acid  to  protein 
the  Cl'  bound  by  the  protein  was  about  20  per  cent  less  than  the 
proportion  of  H+  which  was  bound. 

It  has  been  shown  by  Blasel  and  Matula  (2)  that  deaminized 


NON-DISSOCIABLE  INORGANIC  RADICAL  169 

gelatin,  prepared  by  the  action  of  nitrous  acid  upon  gelatin, 
notwithstanding  the  absence  of  end  NH2  groups,  still  retains  the 
power  of  binding  Cl'  ions  in  solutions  of  hydrochloric  acid. 

Oryng  and  Pauli  (20)  have  shown  that  serum  albumin,  gelatin 
and  deaminized  gelatin,  dissolved  in  solutions  of  potassium  chlo- 
ride, bind  a  definite  proportion  of  Cl'  ions,  and  this  proportion 
is  greatly  increased  by  the  addition  of  acids  (such  as  sulphuric 
acid)  to  the  solution. 

Confirmatory  evidence  is  not  lacking.  Loevenhart  (15)  and 
others  *  have  found  that  rennet  will  not  coagulate  calcium  casein- 
ate  unless  a  small  amount  of  a  dissociable  salt  of  calcium  is  present. 
The  calcium  bound  by  the  casein  itself  is  not  available  for  this 
purpose,  but  if  a  small  quantity  of  acid  be  added  then  a  pro- 
portion of  the  calcium  is  freed  from  its  combination  with  casein 
and,  if  it  forms  a  dissociable  salt  with  the  added  acid,  it  is  able 
to  bring  about  coagulation. 

W.  A.  Osborne  (21)  has  shown  that  if  calcium  caseinate  be 
placed  inside  a  dialysing  tube  which  is  then  immersed  in  a  very 
dilute  solution  of  mercuric  chloride  the  mercury  diffuses  into  the 
tube  and  is  there  held  in  an  undissociated  form,  since  the  con- 
centration of  mercury  within  the  tube  is  found,  after  some  time, 
to  considerably  exceed  that  of  the  mercury  in  the  outside  fluid. 

Similarly,  it  has  been  shown  by  Moore,  Roaf  and  Webster 
(18)  that  if  casein  dissolved  in  dilute  sodium  hydroxide  be  placed 
inside  an  osmometer  of  which  the  membrane  is  permeable  to  crys- 
talloids but  not  to  colloids  and  the  concentration  of  NaOH  be 
rendered  initially  equal  on  both  sides  of  the  membrane,  then 
NaOH  will  move  into  the  osmometer  against  the  osmotic  pressure 
gradient  and  actually  lead  to  a  pronounced  increase  in  the  pres- 
sure within  the  osmometer.  It  is  evident,  therefore,  that  not 
only  OH'  but  also  Na+  ions  must  have  been  bound  by  the  protein, 
since  otherwise  no  movement  of  Na+  across  the  membrane  could 
have  occurred. 

*  The  statement  of  van  Dam  (6).  that  the  extent  of  coagulation  depends 
upon  the  quantity  of  Ca  bound  by  the  casein  is  not  irreconcilable  with  Loeven- 
hart's  results.  As  is  well  known,  the  calcium  bound  in  casein  as  calcium 
caseinate  does  not  suffice  to  bring  about  coagulation.  A  dissociable  salt  of 
calcium  must  also  be  present.  That  this  salt  may  combine  with  the  calcium 
paracaseinate  to  form  a  double  salt  analogous  to  those  described  in  the 
previous  chapters,  is  not  at  all  unlikely. 


170  ELECTROCHEMISTRY 

Rohmann  and  Hirschstein  (30)  have  shown  that  solutions  of 
silver  caseinate  contain  no  silver  ions,  since  they  fail  to  yield  a 
precipitate  on  adding  sodium  chloride. 

The  most  obvious  conclusions  to  be  drawn  from  these  results 
are  (a)  that  the  protein-base  or  protein-acid  compounds  are  not 
subject  to  dissociation  at  all;  or  else  (6)  that  they  dissociate  the 
positive  and  negative  ions  of  the  inorganic  constituent  in  equiva- 
lent proportions,  i.e.,  undergo  hydrolytic  dissociation. 

That  these  assumptions  are  incorrect,  however,  is  shown  by 
a  large  number  of  experiments  which  demonstrate  that  the  pro- 
tein compounds  with  inorganic  bases  and  acids  are  true  electro- 
lytes, independently  of  any  hydrolytic  dissociation  which  they 
may  undergo  in  solution.  For  example,  Sjoqvist  (35)  has  shown 
that  if  egg-albumin  be  dissolved  in  dilute  hydrochloric  acid,  as 
the  concentration  of  albumin  is  increased,  keeping  that  of  the 
HCl-solution  constant,  the  molecular  conductivity  (calculated  for 
0.025  AT  HC1)  diminishes  until  it  reaches  a  constant  minimum 
value,  which  is  attained  when  about  four  grams  are  dissolved 
in  100  cc.  of  0.025  N  HC1.  Now  the  above  quoted  results  of 
Bugarszky  and  Liebermann  show  that  in  this  solution  at  least  97 
per  cent  of  the  hydrochloric  acid  is  bound  by  the  egg-albumin. 
The  observed  " molecular"  conductivity  (67  X  10~3)  is  at  least  7 
times  greater  than  could  be  accounted  for  by  the  maximum 
possible  residuum  of  unneutralized  hydrochloric  acid  and  must 
therefore  be  due  to  the  protein-acid  compound. 

Solutions  of  the  caseinates  of  the  alkalies  and  alkaline  earths 
can  be  obtained  which  are  neutral  or  even  acid  to  litmus  (Cf. 
Chap.  V),  these  solutions  therefore  contain  no  free  base;  never- 
theless they  are  excellent  conductors  of  electricity  (34)  (23)  (27) 
(28)  since  a  2  per  cent  solution  of  potassium  caseinate  which  is 
neutral  to  litmus  possesses  a  conductivity  of  92.7  X  10~3  recip- 
rocal ohms  per  equivalent  of  base  neutralized  at  30°  C.  That 
this  conductivity  is  not  attributable  to  associated  impurities, 
inorganic  or  otherwise,  is  shown  by  the  following  facts: 

(i)  It  bears  a  definite  relation  to  the  amount  of  base  neutralized 
by  the  protein  (27). 

(ii)  The  conduction  of  electricity  is  accompanied  by  migration 
of  the  casein  to  the  anode,  and  the  amount  of  casein  transported 
to  the  anode  is  directly  proportional  to  the  quantity  of  electricity 
which  is  transported  through  the  solution  (29). 


NON-DISSOCIABLE  INORGANIC  RADICAL  171 

Similarly,  solutions  of  the  serum-globulinates  of  the  alkalies 
and  alkaline  earths  may  be  obtained  which  are  neutral  to  litmus 
and  which  nevertheless  conduct  electricity,  the  passage  of  a 
direct  current  through  these  solutions  being  accompanied  by 
transport  of  the  protein  to  the  anode  (10). 

Only  one  conclusion  is  left  open  to  us,  therefore,  namely,  that 
the  salts  which  proteins  form  with  inorganic  acids  and  bases  do 
not  dissociate  at  the  point  of  union  of  the  inorganic  radical  with 
the  protein,  but  elsewhere,  within  the  protein  molecule  itself, 
yielding,  not  an  inorganic  and  a  protein  ion,  but  two  or  more 
protein  ions,  in  one  or  more  of  which  the  inorganic  radical  is 
bound  up  in  a  non-dissociable  form.* 

On  examining  the  details  of  the  behavior  of  the  protein  salts, 
as  electrolytes,  we  are  speedily  compelled,  by  inference,  to  reach 
precisely  the  same  conclusion. 

In  the  first  place,  the  conductivity  of  solutions  of  certain 
protein  salts,  for  example  potassium  caseinate,  is  not  at  all  affected 
by  the  presence,  in  the  solution,  of  an  excess  of  the  ions  of  the 
inorganic  radical.  This  fact  is  very  clearly  shown  by  the  fol- 
lowing experiments  (27). 

Two  and  a  half  grams  of  pure  casein  were  dissolved  in  solu- 
tions containing  varying  amounts  of  KOH  of  which,  in  each 
instance,  so  much  was  neutralized  by  0.1  N  HC1  as  to  leave  the 
equivalent  of  25  cc.  of  0.1  N  KOH  unneutralized  by  the  acid. 
These  solutions  were  then  each  diluted  to  250  cc.,  so  that  the  final 
solutions  consisted  of  1  per  cent  casein  dissolved  in  0.01  N  KOH 
plus  varying  amounts  of  KC1.  The  conductivities  ( =  x)  of  these 
solutions  (at  30  degrees)  were  then  determined  and  also  the 
conductivities  (=  Xi)  of  solutions  similarly  made  up  without  the 
introduction  of  casein.  The  conductivity  of  the  original  solu- 
tion, before  the  introduction  of  casein,  is  the  sum  of  two  quanti- 
ties, i.e.,  the  conductivity  of  0.01  KOH  +  the  conductivity  of 
the  KC1;  that  of  the  solution  of  the  caseinate  is  the  sum  of  three 
quantities,  i.e.,  the  conductivity  of  the  unneutralized  KOH  and 
the  conductivity  of  the  KC1  +  the  conductivity  of  the  caseinate. 
Subtracting  the  latter  from  the  former,  therefore,  we  obtain 
Xi  —  x,  which  is  the  conductivity  of  the  0.01  KOH  minus  the 
conductivity  of  the  KOH  unneutralized  by  1  per  cent  casein 
and  the  conductivity  of  the  casemate  itself;  in  other  words,  the 

*  Cf.  also  Chap.  IX,  3. 


172 


ELECTROCHEMISTRY 


conductivity  of  a  solution  of  the  neutralized  KOH  minus  the 
conductivity  of  the  caseinate.  If  we  subtract  this  quantity 
(xi  —  x)  from  the  conductivity  of  the  neutralized  KOH,  therefore, 
we  obtain  the  conductivity  of  the  caseinate  itself.  Now  in  these 
solutions,  as  determined  by  the  gas-chain,  the  concentration  of 
neutralized  KOH  was  0.00993,  and  the  conductivity  at  30  degrees 
of  a  solution  of  free  KOH  of  this  concentration  would  be  280  X  10~5 
reciprocal  ohms.  Subtracting  Xi  —  x  from  280  X  10~5,  therefore, 
we  obtain  the  conductivity,  in  each  of  the  solutions  investigated, 
of  the  caseinate  itself.  In  the  following  table  some  experimental 
results  are  given.  In  the  first  column  is  given  the  concentration 
of  KC1  which  was  present  in  the  solution  of  caseinate;  in  the 
second  the  conductivity  (=  Xi)  of  the  solution  containing  no 
casein;  in  the  third  the  conductivity  (=  x)  of  the  solution  con- 
taining casein;  in  the  fourth  the  conductivity  (=  280  X  10~5  — 
(xi  —  x))  of  the  caseinate  itself;  in  the  fifth  the  deviation  of  this 
conductivity  from  its  average  value  in  the  different  solutions; 
in  the  sixth  the  experimental  error  of  the  conductivity  meas- 
urement itself,  exclusive  of  any  possible  deviations  arising  out 
of  errors  in  weighing,  measurements  of  volume,  or  decomposition 
of  the  casein. 


Concen- 
tration of 
KC1 

Xi  (recip.  ohms) 

x  (recip.  ohms) 

Conductivity 
of  caseinate  it- 
self 

=  Deviation 
from  average 

=  Experimen- 
tal error 

0.00  AT 

o.oi  AT 

0.02  AT 
0.03  AT 

277.4X10-5 
417.0X10-5 
548.0X10-5 
698.0X10-5 

81.2X10-5 
222.6X10-5 
360.5X10-5 
494.5X10~5 

83.8X10-5 
85.6X10-5 
91.8X10-5 
76.1X10-5 

-0.5X10-5 
+  1.3X10-5 
+7.5X10-5 
-8.2X10-5 

±0.  7X10~5 
±1.8X10-5 
±3.5X10-5 
±5.9X10-6 

It  is  evident  that  the  estimates  of  the  conductivity  of  the 
caseinate  itself  which  were  thus  obtained  are  appreciably  con- 
stant, the  deviation  from  the  average  being  irregular  and  less  or 
only  very  slightly  greater  than  the  purely  metrical  error  of  the 
observation.  In  other  words,  whether  the  concentration  of  KC1 
in  its  solution  is  0.00  or  0.03  the  share  taken  by  the  potassium 
caseinate  itself  in  conducting  the  current  is  the  same  within  the 
experimental  error,  that  is,  at  the  very  highest  estimate,  within 
10  per  cent. 

Now  suppose  that  potassium  caseinate,  containing  this  pro- 
portion of  potassium,  yields,  on  dissociation,  only  one  potassium 


NON-DISSOCIABLE  INORGANIC  RADICAL  173 

ion  and  one  casein  ion,  then  applying  the  ordinary  laws  of  dis- 
sociation, since  in  the  solution  containing  no  KC1  the  total  con- 
centration of  K  ions  must  have  been  very  nearly  0.01  and  calling 
A  the  concentration  of  protein  ions  in  this  solution,  C  the  dissoci- 
ation-constant of  the  caseinate  and  X  the  concentration  of  the 
undissociated  casemate  we  have,  very  nearly: 

0.01  A  =  KX 

while  the  solution  containing  0.04  N  potassium  (0.03  N  KC1)  we 
have,  very  nearly, 


whence  A  =  26  X  ,  or  the  caseinate  must,  in  the  first  solution,  have 
been  f  f  ths  dissociated.  Now  it  should  be  particularly  observed 
that  this  is  most  decidedly  a  minimal  estimate  of  the  degree  of 
dissociation  of  this  salt,  if  the  assumption  upon  which  we  have 
proceeded  is  correct.  For  free  casein  is  insoluble  and  1  gram  of 
casein  is  just  carried  into  solution  by  11.4  X  10~5  equivalents 
of  base,  whereas  these  solutions  contained  as  we  have  seen  nearly 
nine  times  this  amount  of  neutralized  KOH.  If  we  suppose  that, 
in  reality,  nine  COOH  groups  in  the  casein  molecule  have  been 
neutralized  by  KOH  in  these  solutions,  so  that  the  caseinate 
yields  nine  K+  ions,  then  the  above  solution  (i.e.,  that  which 
contained  no  KC1)  must  have  been  no  less  than  fMffffths 
dissociated.  Hence,  at  the  very  lowest  estimate,  if  we  assume 
that  it  dissociates  potassium  ions,  a  solution  of  potassium  casein- 
ate  containing  99  X  10~5  equivalents  of  potassium  per  gram  of 
caseinate  must  be  f  £ths  dissociated. 

Upon  dilution,  therefore,  its  conductivity  could  only  increase 
by  sVth,  or  4  per  cent  and,  moreover,  no  solution  of  potassium 
caseinate  could  be  possessed  of  a  greater  equivalent  conductivity 
than  that  of  the  above  solution  plus  4  per  cent,  i.e.,  about  90  X  10~3 
at  30°  C. 

But,  as  we  shall  see  in  succeeding  chapters,  the  equivalent  con- 
ductivity of  a  1  per  cent  solution  of  caseinate  in  0.01  N  KOH 
increases  very  much  more  than  4  per  cent  on  dilution,  nor  can 
this  increase,  as  we  shall  see,  be  attributed  to  hydrolytic  dissocia- 
tion. Moreover  solutions  of  potassium  caseinate  can  readily  be 
obtained  which  are  add  to  litmus  and  which  possess  an  equivalent 


1 74  ELECTROCHEMISTRY 

conductivity  (calculated  upon  the  basis  of  the  potassium  which 
they  contain)  of  over  120  X  10~3  or  30  per  cent  more  than  that 
of  the  solution  considered  above. 

Hence  we  have  no  resource  but  to  conclude  that  our  initial 
assumption  was  erroneous  and  that  potassium  caseinate  dis- 
solved in  water  does  not  yield  potassium  ions.* 

Again,  we  may  reach  the  same  conclusion  by  quite  a  different 
process  of  reasoning  and  from  very  different  experimental  data. 
The  conductivity  of  solutions  of  the  caseinates  and  globulinates 
of  the  alkalies  and  alkaline  earths  and  of  the  salts  which  ovo- 
mucoid  forms  with  acids  does  not  decrease  in  direct  proportion 
with  the  dilution,  but  more  slowly,  indicating  a  progressively 
increasing  dissociation  of  the  caseinate  on  dilution.  From  the 
curve  expressing  the  relation  between  the  equivalent-conduc- 
tivity (calculated  on  the  basis  of  the  inorganic  radical)  and  the 
dilution  we  can,  by  exterpolation,  estimate  the  maximum  equiva- 
lent conductivity,  i.e.,  the  equivalent  conductivity  at  infinite 
dilution  of  the  salt;  this  we  can  do  rather  accurately,  since  at 
readily  attainable  dilutions  the  equivalent  conductivity  already 
increases  very  slowly  with  dilution  and  obviously  tends  to  ap- 
proach a  constant  maximum.  Now,  as  is  well  known,  this  maxi- 
mum bears  a  constant  proportion  to  the  sum  of  the  equivalent 
conductivities  of  the  ions  into  which  the  salt  dissociates. 

If  the  inorganic  radical  is  dissociated  as  such,  the  equivalent 
conductivity  of  these  salts  cannot  be  less  than  that  of  the  inor- 
ganic radical  itself,  but  must  exceed  it  by  a  quantity  equal  to 
the  equivalent  conductivity  of  the  protein  ion  or  ions.  In  the 
following  table  are  compared  the  observed  equivalent  conduc- 
tivities (at  infinite  dilution)  of  a  number  of  protein  salts  at  30 
degrees, f  and  those  of  the  inorganic  radicals  which  they  contain, 
calculated  from  the  data  given  by  Kohlrausch  and  Holborn  (12)4 

*  It  may  be  mentioned  in  passing,  that  in  the  solutions  investigated 
potassium  caseinate  evidently  does  not  form  a  double  salt  with  potassium 
chloride.  Were  such  a  double  salt  formed,  and  we  have  seen  that  in  many 
cases  salts  of  this  type  may  be  formed  in  protein  solutions,  then,  of  course,  the 
KC1  would  not  be  without  effect  upon  the  conductivity  of  the  caseinate. 

t  Cf.  data  cited  in  Chap.  X. 

t  The  migration-velocities  of  the  ions  at  18  degrees  (p.  200)  increased  by 
2  per  cent  per  degree  to  reduce  to  the  temperature  employed  and  multiplied 
by  the  proportionality  between  ionic  velocity  and  reciprocal  ohms  per  cc.,  i.e., 
96.44. 


NON-DISSOCIABLE  INORGANIC  RADICAL 


175 


Salt 

Temp., 
Degrees 
C. 

Equivalent  con- 
ductivity at  in- 
finite dilution, 
equivalent  cone, 
taken  as  that  of  the 
inorganic  radical. 
Reciprocal  ohms 
per  cc.  per  equiva- 
lent per  litre 

Equivalent  con- 
ductivity at  in- 
finite dilution  of 
the  inorganic  radi- 
cal.    Reciprocal 
ohms  per  cc.  per 
equivalent  per 
litre 

Sodium  caseinate  (80X10"5  equivs. 
per  gram) 

25 

63  5X10~3 

50  6X10~3 

Ammonium  caseinate  (80X10~5 
equivs.  per  gram) 

25 

79  4X10~3 

73  2X10-3 

Potassium  caseinate  (80X10~5 
equivs.  per  gram)  .   . 

30 

80.6X10~3 

81.0X10-3 

Calcium  caseinate  (80X10~5  equivs. 
per  gram)  

30 

35.9X10~3 

65.7X10-3 

Strontium  caseinate  (80X10~5 
equivs.  per  gram)  

30 

30.7X10-3 

67.0X10-3     . 

Barium  caseinate  (80X10"5  equivs. 
per  gram) 

30 

42  1X10-3 

71  1X10-3 

Potassium  serum-globulinate 
(20X10~5  equivs.  per  gram) 

30 

51  OX1Q-3 

81  0X10-3 

Calcium  serum-globulinate 
(20XlO~5  equivs.  per  gram). 

30 

23.5X10-3 

65.7X10-3 

Strontium  serum-globulinate 
(20X10"5  equivs.  per  gram)  
Barium  serum-globulinate 
(20X10~5  equivs.  per  gram)  
Ovomucoid  chloride  (45X10~5 
equivs.  per  gram) 

30 
30 
30 

27.5X10-3 
23.4X10-3 
196  0X10-3 

67.X  1C-3 
71.1X10-3 
81  7X10-3 

It  is  evident  that  in  many  cases  the  equivalent  conductivity 
of  a  protein  salt  is  actually  less,  in  the  salts  of  the  alkaline  earths 
very  considerably  less  than  that  of  its  inorganic  radical  alone. 
On  the  supposition  that  the  protein  salt  splits  off  the  inorganic 
radical  as  an  ion,  not  only  the  inorganic  radical  but  also  the 
protein  must  be  participating  in  the  conduction  of  electricity 
through  its  solution,  and  its  equivalent  conductivity,  when  com- 
pletely dissociated,  must  be  greater  than  that  of  the  inorganic 
radical  by  the  amount  contributed  by  the  protein  ion.  Hence 
the  assumption  that  the  salts  of  the  proteins  split  off  the  inorganic 
radical  as  an  ion  must  be  incorrect. 

We  have  seen,  in  considering  the  constitution  of  the  protein 
molecule  (Chap.  I),  that  the  protein  molecule  does  not  contain 
a  sufficient  number  of  terminal  —  NH2  and  —  COOH  groups  to 
account  for  its  high  combining  capacity  for  acids  and  bases,  and 
the  suggestion  was  put  forward  that  the  true  point  of  union  with 
acids  and  bases  is  the  —  N.HOC—  group,  and  that  union  with 


176  ELECTROCHEMISTRY 

bases  and  acids  and  the  dissociation  of  the  resultant  salts  take 
place  according  to  the  following  schemes: 

H 

I          ++ 

-N.HOC-  -|-  NaOH  =  -N"  +  NaOC- 

I 
OH 

H 

I          ++ 
-N.HOC-  +  HC1  =  -N"  +  HOC- 

I 
Cl. 

or  in  accordance  with  modifications  of  these  schemes  arising  out 
of  the  participation  of  dicarboxylic-  and  diamino-radicals  in  the 
reactions. 

In  the  succeeding  pages  the  endeavor  will  be  made  to  interpret 
the  electrochemical  behavior  of  the  proteins  and  their  salts  with 
the  aid  of  this  hypothesis. 

2.  The  Electrolysis  of  Protein  Salts.  —  It  was  shown  by 
Hardy  (9)  in  1899  that  if  a  trace  of  acid  be  added  to  a  solution 
of  dialysed  white  of  egg,  modified  by  dilution  and  boiling,  on 
passing  a  direct  current  through  the  solution  the  whole  of  the 
protein  finally  moves  over  to  the  cathode,  where  it  is  precipitated. 
If  a  trace  of  alkali  is  added,  however,  instead  of  acid,  the  whole 
of  the  protein  finally  migrates,  under  the  influence  of  the  current, 
to  the  anode.  He  later  showed  that  serum-globulin  in  solution 
behaves  similarly  (10).  Under  such  circumstances,  therefore,  the 
protein  behaves  like  the  anion  or  cation  of  a  salt;  like  the  cation 
when  combined  with  an  acid;  like  the  anion  when  combined 
with  a  base. 

Here  an  apparent,  but  not  a  real  difficulty  confronts  us  in  the 
application  of  the  hypothesis  outlined  above  to  the  electrolysis 
of  protein  solutions.  On  the  basis  of  this  hypothesis  the  protein 
should  migrate  in  both  directions,  the  cation,  when  the  protein 
is  combined  with  a  base,  and  the  anion,  when  the  protein  is  com- 
bined with  an  acid,  carrying  the  inorganic  constituent  with  it. 
At  first  sight  it  might  appear  as  if  the  protein  should  be  deposited 
at  both  electrodes,  but  not  when  we  look  more  closely  into  the 
matter.  In  both  of  the  cases  just  cited  the  free,  uncombined 


ELECTROLYSIS  177 

protein  is  insoluble,  while  the  combined  protein  is  soluble.  Con- 
sider the  electrolysis  of  a  compound  of  such  a  protein  with  a  base. 

The  anion 

rl 

I 

R.N" 
I 
OH 

will  migrate  to  the  anode,  and  after  neutralizing  any  excess  of 
base  which  may  be  present  (since  the  ion,  obviously,  must  contain 
— N.HOC—  groups  and  therefore,  like  the  entire  molecule,  is  am- 
photeric)  must  eventually,  when  the  film  in  immediate  contact  with 
the  electrode  has  become  saturated  with  protein,  be  precipitated 

as  the  uncombined  and  therefore  insoluble  protein.     The  case  is 

++ 
very  different  with  the  cation  R.CONa,  for  this,  on  arriving  at 

the  cathode,  will  bring  it  an  excess  of  base  and  the  cathode  region 
must  become  alkaline  and,  therefore,  free  protein  cannot  be  pre- 
cipitated there.  Similarly,  in  the  electrolysis  of  a  salt  of  such  a 
protein  with  an  acid  the  uncombined  protein  must  be  precipi- 
tated at  the  cathode,  but  not  at  the  anode.  If  the  protein  is 
of  such  a  type  that  the  free  protein  is  soluble,  for  example  ovo- 
mucoid,  I  have  observed  that  no  deposition  of  protein  occurs  at 
either  electrode,  but  only  the  evolution  of  gas,  presumably  hydro- 
gen at  the  cathode  and  oxygen  at  the  anode.  That  under  such 
conditions  the  protein  nevertheless  migrates  to  both  electrodes  has 
been  shown  by  Stirling  and  Brito  (36)  and  by  Howell  (11). 

If  a  direct  current  of  about  one  milliampere  be  passed  through 
a  solution  of  potassium  caseinate,  which  is  neutral  to  litmus, 
gas  may  be  observed  to  be  evolved  at  both  electrodes,  but  a  firm 
white  spongy  precipitate  is  deposited  upon  the  anode,  the  cellu- 
lar texture  of  which  is  attributable  to  entangled  bubbles  of  gas, 
presumably  oxygen  (29). 

A  quantity  of  this  precipitate  was  collected.  The  anode  con- 
sisted of  a  spiral  of  platinum  wire  some  9  cm.  long.  This  wire, 
when  coated  with  the  precipitate,  was  well  washed  in  a  stream 
of  distilled  water,  and  the  precipitate  was  then  scraped  off  into 
99.8  per  cent  alcohol.  The  precipitate  which  was  thus  collected 
under  alcohol  was  washed  with  alcohol  and  ether  and  dried  at 
30  degrees  over  sulphuric  acid  for  48  hours. 

The  precipitate  proved  to  be  uncombined  (base  free)  casein. 


1 78  ELECTROCHEMISTRY 

In  fact  so  nearly  was  it  devoid  of  mineral  content  as  to  practi- 
cally realize  that  elusive  ideal,  the  " ash-free  protein,"  for  one 
gram  of  the  precipitate  yielded  less  than  two  milligrams  of  ash. 
Yet  this  casein,  in  every  way  in  which  it  was  tested,  proved  to 
be  perfectly  normal.  The  possibility  was  thus  indicated  of  esti- 
mating the  electrochemical  equivalent  of  casein. 

Solutions  of  potassium  caseinate  were  placed  in  a  U-tube  of 
about  30  cc.  capacity,  25  cc.  of  solution  being  employed  in  each 
experiment.  The  anode  consisted  of  a  spiral  of  platinum  wire 
about  \  mm.  thick  and  9  cm.  long,  the  diameter  of  the  spiral 
being  about  \  cm.  and  its  " pitch"  about  45  degrees.  The  cathode 
simply  consisted  of  a  platinum  wire  dipped  in  the  fluid  in  the 
other  arm  of  the  U-tube.  The  U-tube  was  provided  at  the 
bottom  with  a  3-way  stop-cock,  which  could  either  be  turned  so 
as  to  provide  fluid  communication  between  the  two  arms  of  the 
tube,  or  else  so  as  to  permit  the  contents  of  the  anodal  limb  to 
escape  into  a  receptacle.  In  this  way  it  was  possible  to  investi- 
gate separately,  if  desired,  the  contents  of  each  arm  of  the  U-tube. 
It  was  found  that  after  electrolysis  the  fluid  in  the  anodal  arm, 
in  which  the  deposition  of  casein  occurred,  was  practically  un- 
altered in  reaction,  although  its  casein-content  was  much  dimin- 
ished. In  the  cathodal  arm,  not  only  did  the  casein-content 
diminish,  but  the  alkalinity  of  the  fluid  was  markedly  increased. 

The  current  was  led  from  the  terminals  of  a  110-volt  circuit 
through  a  16  candle-power  lamp,  a  milliamperemeter,  a  silver 
titration  voltameter  containing  30  cc.  of  JV/100  AgN03,  and 
through  the  solution  of  caseinate. 

The  silver  was  then  determined  by  titration  with  a  AT/100 
ammonium  thiocyanate  in  the  presence  of  a  constant  excess  of 
nitric  acid,  a  constant  quantity  (5  cc.  of  saturated  solution}  of 
ferric  alum  being  employed  as  indicator. 

The  amount  of  casein  which  had  been  precipitated  by  the 
current  was  estimated  by  determining  the  refractive  indices  of 
the  original  and  of  the  electrolysed  solutions  at  the  same  tem- 
perature; the  differences  between  the  refractive  indices  divided 
by  0.00152  yielding  the  decrease  in  the  percentage  of  casein  con- 
tained in  the  solution  consequent  upon  electrolysis.*  The  quan- 
tity of  solution  employed  was  always  25  cc.  Hence  the  decrease 

*  Cf.  Chap.  XIV.    Also  T.  Brailsford  Robertson  (24). 


ELECTROLYSIS  179 

in  the  percentage-content  of  casein,  divided  by  4,  was  the  amount 
of  casein  precipitated  by  the  current. 

The  experiments  were  all  conducted  at  30°  C. 

Varying  amounts  of  casein  were  dissolved  in  100  cc.  each  of 
KOH  solutions  of  varying  concentrations  so  that  the  proportion 
of  base  to  casein  was  50  X  10~5  equivalents  per  gram  (neutral 
to  litmus),*  or  80  X  10~5  equivalents  per  gram  (neutral  to  phenol- 
phthalein),f  or  100  X  10~5  equivalents  per  gram.  In  estimating 
the  current  employed,  the  electrochemical  equivalent  of  silver, 
in  grams  per  coulomb,  is  taken  as  0.001118  (8). 

It  was  found  that  the  solutions  containing  the  higher  pro- 
portions of  base  to  casein  yielded  an  apparently  lower  electro- 
chemical equivalent  for  the  casein.  This  was  speedily  traced  to 
resolution  of  the  casein  from  the  electrode  after  precipitation. 
The  anode,  after  having  been  coated  with  casein  by  the  action 
of  a  current  of  one  milliampere  passing  through  a  3  per  cent 
solution  of  casein,  neutral  to  litmus,  was  washed  in  water,  alcohol 
and  ether,  dried  and  weighed.  It  was  then  immersed  in  a  solu- 
tion of  casein  (3  per  cent)  neutral  to  litmus  for  2  hours,  then 
withdrawn  and  washed,  dried  and  weighed  as  before.  It  was 
found  to  have  lost  11  milligrams  in  weight.  Similar  experiments 
were  conducted  in  which  the  solutions  in  which  the  coated  wire 
was  immersed  were  neutral  to  phenolphthalein  and  alkaline  to 
phenolphthalein.  The  following  were  the  results  obtained: 


Current  em- 
ployed for  pre- 
cipitation 

Solution 

Loss  after 
two  hours 

1  milliamp. 
1  milliamp. 
2  milliamp. 
1  milliamp. 

3  per  cent  casein  in 
3  per  cent  casein  in 
3  per  cent  casein  in 
3  per  cent  casein  in 

50XKr5equivs. 
80XlO~5equivs. 
80X10~5equivs. 
100XlO~5equivs. 

KOH  grm. 
KOH  grm. 
KOH  grm. 
KOH  grm. 

11  mg. 
54  mg. 
59  mg. 
97  mg. 

Hence  doubling  the  rate  of  deposition  makes  very  little,  if 
any,  difference  to  the  rate  of  resolution  of  the  casein,  but  increas- 
ing alkalinity  of  the  solution  in  which  the  deposition  occurs 
increases  the  rate  of  resolution  very  markedly.! 

*  Cf.  Chap.  V.    Also  T.  Brailsford  Robertson  (27). 

t  Cf.  Chap.  V. 

$  If  the  anode  be  much  less  than  9  cm.  in  length  there  is  a  tendency,  after 
prolonged  electrolysis,  to  what  may  be  termed  "flocculent  deposition,"  or 
precipitation  of  the  casern  within  the  body  of  the  fluid  in  the  anodal  arm  and 


180  ELECTROCHEMISTRY 

Assuming  the  rate  of  resolution  to  be  constant,  since  the  con- 
tents of  the  anodic  arm  are  not  appreciably  altered  in  reaction 
in  the  electrolysis,  it  is  possible  to  calculate  from  the  above  data 
for  each  of  the  solutions  and  periods  employed  the  loss  due  to 
resolution.  In  the  column  headed  "Loss  due  to  resolution"  in 
the  accompanying  tables  these  quantities  are  given.  On  adding 
them  to  the  amounts  of  casein  lost  from  the  solution  from  which 
the  "apparent"  values  of  the  electrochemical  equivalent  are 
estimated  one  obtains  the  "corrected"  values  corresponding  to 
the  total  precipitation  actually  induced  by  the  passage  of  the 
current. 

The  possible  error  in  the  refractometer  reading  is  1'  of  the 
angle  of  total  reflection,  this  corresponds  to  an  error  of,  ±0.00010  in 
the  refractive  index,  that  is,  to  an  error  of  ±0.07  in  the  estimated 
decrease  in  the  percentage  of  casein  due  to  electrolysis  of  the 
solution,  and  to  an  error  of  ±0.0175  in  the  estimate  of  the  amount 
of  casein  deposited  by  the  current.  The  possible  error  in  each 
estimate  of  the  electrochemical  equivalent,  arising  from  this  source, 
is  indicated  in  the  tabulated  results. 

The  average  values  for  the  electrochemical  equivalent  in  the 
different  solutions  are  obviously,  within  the  experimental  error, 
identical.  Now  in  solutions  containing  50  X  10~5  equivalents 
of  base  per  gram,  the  combining  capacity  of  casein  is  50  X  10~5 
equivalents  per  gram,  in  solutions  containing  80  X  10~5  equiva- 
lents per  gram,  it  is  80  X  10~5  equivalents  per  gram,  and  in  solu- 
tions containing  100  X  10~5  equivalents  per  gram  it  is  between 

99  and    100  X  10~5   equivalents   per   gram.*     In   the   solutions 
investigated,  therefore,  the  combining  weight  of  the  casein  varies 

100  per  cent,  yet  the  electrochemical  equivalent,  measured  in  this 
way,  remains  the  same. 

These  facts  are  to  be  interpreted  as  follows :  We  have  seen  that 
the  casein  anion,  which  is  free  from  base,  must  migrate  to  the 
anode.  There  it  may  be  presumed  to  react  with  water,  liber- 
ating oxygen  and  free  casein,  which  combines  with  the  excess 

not  upon  the  wire.  Under  these  conditions,  resolution  is  apparently  more 
rapid  and  even  if  the  flocculent  deposit  be  filtered  off  from  the  anodal  fluid 
before  the  alkaline  cathodal  fluid  is  mixed  with  it,  the  values  of  the  electro- 
chemical equivalent  are  low.  No  such  phenomenon  was  observed  when  the 
anode  was  of  sufficient  length. 

*  Cf.  Chap.  IX.     Also  T.  Brailsford  Robertson  (27). 


ELECTROLYSIS 


181 


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§ 


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fi-8 


8888888 

ddddddo 


O  CO  (N  CO  -^  (N  <M 

ddddddo 
-H  -H  -H  -H  -H  -H  -H 


o 


1C  iO  iO  1C  »C  1C  »O 

poppopp 
ddddddo 
-H -H -« -H -H -H -H 

iO  10  *O  O  iC  O  OO 
i— I  CO  OO  OO  i— I  OO  CO 

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ddddddo 


S  S.S.S.S.S.S 
S  S  p  B  S  S  9 
»oojco  »o 

02    02    CO    02    GQ    02 

(N  <N  (M  <N  (M  (N  T-H 


OOOOOOO 

XXXXXXX 


T-H  O5  00  O  I^  T-H  10 


CO  Tt<  •*  CO  CO  <N  C<l 


182 


ELECTROCHEMISTRY 


98 


X 


II 

0.0" 


C<1  lO  1C  ^  CO  CO  to 

ddddddd 
-H -H -H -H -H -H -H 


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T— I    1-H    T— (    1— I    C^    C? 

ddddddd 

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o  i— 1 1^  co  o  oo  o 

O5  l>  »O  O5  tO  CO  OO 

ppppopp 
ddddddd 


ppppopp 
ddddddd 
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O  CO  O  OO  CO  <N  »O 

OO  l>-  T— (  CD  !>•  lO  1—* 
-*  O5  00  •*  Oi  O  CO 


ooooooo 


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CO  <N 

ELECTROLYSIS  183 

of  base  until  the  proportion  of  base  to  casein  in  the  film  in  im- 
mediate contact  with  the  anode  falls  to  that  which  obtains  at 
"saturation"  of  the  base  with  casein.  Any  additional  casein 
thus  migrating  into  the  film  in  contact  with  the  anode  must  be 
precipitated  as  uncombined  casein.  The  cations,  containing  the 
potassium,  migrate  to  the  cathode  and  there  react  with  water, 
liberating  KOH,  casein  and  hydrogen,  the  casein  reacting  with 
the  excess  of  KOH  to  again  form  potassium  caseinate  and  to 
again  participate  in  carrying  the  current  in  each  direction. 

Hence  the  electrochemical  equivalent  which  is  actually  meas- 
ured in  solutions  of  all  reactions  is  that  of  casein  at  "saturation" 
of  the  base  with  the  protein. 

Rejecting  the  data  obtained  in  the  solutions  alkaline  to  phenol- 
phthalein,  on  account  of  the  possible  error  arising  from  hydro- 
lytic  decomposition  of  the  protein  due  to  the  excess  of  alkali, 
the  average  of  all  the  determinations  yields  the  value  0.0242  ± 
0.0019  for  the  electrochemical  equivalent  of  casein. 

Multiplying  this  by  the  Faraday  constant,  96,530,  we  obtain 
the  weight  of  casein  in  grams  which  transports  one  atomic  charge. 
This  is  2336  =t  183. 

Now  at  "saturation"  of  a  base  by  casein,  the  proportion  of 
base  to  casein  is  11.4  X  10~5  equivalents  per  gram,*  correspond- 
ing, if  at  this  reaction  casein  combines  with  only  one  molecule 
of  base,  with  the  molecular  weight  of  8772.  If  we  assume  that 
at  "saturation"  of  the  base  with  casein  two,  three,  or  four,  etc., 
molecules  of  base  are  bound  up  in  one  molecule  of  caseinate  the 
molecular  weight  of  the  casein  would  be  two,  three,  or  four,  etc., 
times  8772.  Either  of  two  assumptions  may  now  be  made: 

(i)  The  potassium  caseinate  dissociates  into  potassium  and 
casein  ions.  If  this  be  the  case  then  the  weight  of  the  casein 
anion  must  be  that  of  the  molecule  of  casein,  i.e.,  a  multiple  of 
8772,  and  the  valency  of  the  casein  ions  must  be  a  multiple  of 

8772 
2336  ±  183 

i.e.,  of  3.8  dz  0.3,  or,  in  round  numbers,  4. 

(ii)  The  potassium  caseinate  dissociates  into  two  protein  ions 
of  approximately  equal  weight.  If  this  be  the  case  then  the 
weight  of  the  casein  anion  must  be  half  that  of  the  molecule  of 

*  Cf.  Chap.  V.    Also  T.  Brailsford  Robertson  (24)  (27). 


184  ELECTROCHEMISTRY 

casein,  i.e.,  a  multiple  of  4386,  and  the  valency  of  the  casein  ions 

must  be  a  multiple  of  g»,  i-e->  °f  1-9  ±  0-15,  or,  in  round 

numbers,  2. 

Since,  as  we  have  seen  in  the  earlier  part  of  this  chapter,  the 
former  of  these  two  assumptions  is  inadmissible,  we  may  con- 
clude that  the  valency  of  the  casein  ions,  in  solutions  of  a  base 
11  saturated"  with  casein,  is  a  multiple  of  2.  This  obviously  cor- 
responds with  the  view  that  the  caseinate  dissociates  into  two 
protein  ions  in  accordance  with  the  scheme: 

H 

I          ++ 

Ri.N  =  C.R2  +  KOH  =  RjN"  +  KOCR* 
I  i 

OH  OH 

3.   The  Relative  Masses  of  Protein  Anions  and  Cations.  — 

From  the  above  experimental  results  it  appears  that  the  masses 
of  the  protein  cation  and  anion  must  be  nearly  equal,  for  other- 
wise the  weight  of  one  ion  would  not  be  one-half  but  some  other 
fraction  of  the  entire  molecule  and  the  valency,  deduced  from 
the  above  experimental  data,  would  not  be  a  whole  number  but 
some  fraction.  Since  the  valency  of  an  ion  is  necessarily  a  whole 
number  and  not  a  fraction,  the  weights  of  the  cations  and  anions 
which  the  protein  molecule  yields  must  be  nearly  equal.  The 
above  data  do  not  enable  us,  however,  to  decide  whether  or  not 
the  weights  of  the  cations  and  anions  are  exactly  equal,  since  the 
precision  attained  in  these  experiments  is  not  sufficient  to  reveal 
with  certainty  a  difference  of  less  than  10  per  cent  between  the 
weights  of  the  two  ions. 

It  has  been  shown  by  Bredig  (3)  that  the  equivalent  migration- 
velocities  *  of  very  heavy  ions  under  unit  potential  gradient  at 
constant  temperature  tend  to  approach  a  minimal  constant  value 
of  about  20  X  10~5  cm.  per  sec.  at  18°  C.  The  conception  de- 
veloped above,  therefore,  of  the  mode  of  dissociation  of  the  salts 
of  a  protein  leads  to  the  conclusion  that  the  velocities  of  migra- 
tions of  both  the  cation  and  the  anion  of  a  protein  salt  must  be 

*  That  is,  the  migration-velocity  under  unit  force.  For  a  divalent  ion 
under  unit  potential  gradient  the  force  exerted  is  twice  as  great  as  that  which 
is  exerted  on  a  univalent  ion;  the  absolute  velocity  is,  therefore,  twice  as  great 
as  that  of  a  univalent  ion,  but  the  equivalent  velocity  is  the  same. 


RELATIVE  MASSES  OF  PROTEIN  IONS 


185 


equal.  Equal  numbers  of  protein  ions  must  therefore  migrate 
to  the  anode  and  to  the  cathode  respectively.  Now  since  only 
one  of  these  ions  is  precipitated  it  would  appear  possible  to  de- 
termine whether  or  not  they  are  of  equal  weight  by  measuring 
the  change  in  concentration  at  the  anodal  and  cathodal  regions. 
Employing  Hittorf  s  method  of  representation,  in  the  accompany- 
ing diagram  let  the  region  to  the  right  represent  the  cathodal  and 
that  to  the  left  the  anodal  region.  Let  the  black  spots  repre- 
sent anions  and  the  white  cations.  The  initial  state  of  the  solu- 
tion is  represented  by  the  first  double  row  of  spots  and  its  final 
condition,  after  the  decomposition  of  six  molecules  by  the  current, 
by  the  second  double  row  of  spots. 


o 

o 

o 

0 

O 

o 

o 

o 

o 

0 

o 

o 

O 

o 

o 

o 

o 

o 

o 

o 

0 

o 

0 

© 

Since  the  velocities  of  the  ions  are  presumed  to  be  equal,  the 
number  of  undecomposed  molecules  of  the  original  caseinate 
must,  after  the  passage  of  the  current,  be  the  same  on  each  side. 
The  ions  which  have  separated  to  the  left  (anions)  have  been 
precipitated  while  those  which  have  migrated  to  the  right  have 
remained  in  solution.  It  is  clear  from  the  above  diagram  that 
on  the  right  six  anions  have  been  lost  and  six  cations  have  been 
gained.  If  they  were  equal  in  weight,  therefore,  it  would  appear 
as  if  the  concentration  of  casein  in  the  right  (cathodal)  half  should 
remain  unaltered  by  the  passage  of  electricity.  A  moment's  con- 
sideration, however,  will  suffice  to  show  that  this  is  not  correct. 
In  attaining  this  conclusion  the  assumption  has  been  made  that 
the  casein  cations  after  reaching  the  cathode,  no  longer  partici- 
pate in  the  carrying  of  current.  Unquestionably  this  is  not  the 
case.  As  we  shall  see  in  the  two  succeeding  chapters,  the  com- 
bining capacity  of  casein  increases  markedly  with  increasing 


186  ELECTROCHEMISTRY 

alkalinity  of  its  solution,  at  first  in  direct  proportion,  each  fresh 
equivalent  of  combined  alkali  splitting  another  —  N.HOC—  union. 
Therefore  the  potassium  or  other  metal  which  is  transported 
along  with  the  casein  to  the  cathode,  on  being  transformed  there 
into  the  hydrate,  will  split  the  ion  with  which  it  has  travelled 
into  two,  thus: 

+  2  H2O 
=  H2N.Ri.COH.NR2.COOH  +  KOH  +  H2 

+  KOH  H 

=  H2N.R1.COK+  +  ^N.R2.COOH 
I 
OH 

the  resultant  cation  may  be  presumed  to  be  retained  in  the  cath- 
ode region  while  the  anion  migrates  to  the  anodal  region.  Each 
cation  which  the  cathodal  region  gains,  therefore,  yields  up, 
provided  the  masses  of  the  cations  and  anions  are  in  every  case 
equal,  one-half  of  its  mass  to  the  anodal  region  again.  Referring 
to  the  diagram  again,  therefore,  the  true  gain  by  the  cathodal 
region  will  be  only  half  of  that  represented,  equal  in  weight  to 
only  three  of  the  six  ions  which  have  been  deposited  on  the  anode, 
while  its  loss  will  have  been  three  molecules,  together  equal  to 
the  total  weight  of  casein  deposited  on  the  anode.  Provided, 
therefore,  the  anions  and  cations  in  a  solution  of  potassium 
caseinate  are  equal  in  weight,  no  matter  what  their  absolute 
weight,  then  it  is  clear  that  the  loss  of  casein  from  the  cathodal 
region  must  be  exactly  half  the  loss  from  the  anodal  region. 

I  have  endeavored  to  test  this  theoretical  conclusion  by  de- 
termining the  loss  of  casein  from  the  anodal  and  cathodal  limbs 
of  the  U-tube  employed  in  the  experiments  described  in  the 
preceding  section  of  this  chapter.  The  following  are  some  ex- 
perimental results.  The  measurements  of  the  amounts  of  casein 
were  made  by  means  of  the  refractometer,  as  described  in  the 
preceding  section. 

(1)   Solution  of  3.75  per  cent  casein  in  0.03  N  KOH;  neutral  to  phenol- 

phthalein. 

Electrolysis  for  2  hours  at  30  degrees.    Current  approx.  1  milliampere. 
Gram  of  casein  lost  from  the  anodal  arm  0  .  062  ±  0  .  007 

Gram  of  casein  lost  from  the  cathodal  arm  0.036  ±0.009 


Ratio  1.9  ±0.7 


MIGRATION-VELOCITY  187 

(2)  Solution  of  4  per  cent  casein  in  0.02  N  KOH;  neutral  to  litmus. 
Electrolysis  for  2  hours  at  30  degrees.     Current  approx.  1  milliampere. 

Gram  of  casein  lost  from  the  anodal  arm  0 . 093  ±  0 . 008 

Gram  of  casein  lost  from  the  cathodal  arm  0 . 045  ±  0 . 007 

Ratio  2. 15  ±0.55 

(3)  Solution  of  3.75  per  cent  casein  in  0.03  N  KOH;  neutral  to  phenol- 

phthalein. 

Electrolysis  for  2  hours  at  30  degrees.     Current  approx.  1  milliampere. 
Gram  of  casein  lost  from  the  anodal  arm  0 . 070  db  0 . 008 

Gram  of  casein  lost  from  the  cathodal  arm          0 . 045  ±  0 . 009 

Ratio  1 . 65  ±  0 . 55 

Within  the  experimental  error,  therefore,  the  ratio  of  the  anodal 
to  the  cathodal  loss  is  2,  as  demanded  by  theory.  The  experi- 
mental error  in  determining  the  ratio  is,  however,  large,  as  the 
above  figures  reveal,  and  although  these  results  may  be  taken  as 
confirmatory  of  the  general  correctness  of  the  above  outline  of 
the  mechanism  of  electrolysis  in  these  solutions,  yet  they  do  not 
suffice  to  enable  us  to  determine  whether  or  not  the  protein  anions 
and  cations  are  absolutely  equal  in  mass.  Further  elaboration 
and  refinement  in  the  technique  of  these  measurements  will 
doubtless  enable  us  in  the  future,  however,  to  measure  the  rela- 
tive masses  of  the  protein  anions  and  cations  with  considerable 
precision. 

In  passing  it  may  be  pointed  out  that  these  results  afford  a 
striking  confirmation  of  the  view  which  I  have  developed  above 
that  the  protein  salts  in  solution  in  water  do  not  yield  protein 
and  inorganic  ions  but  only  protein  ions.  Referring  to  the 
Hittorf  diagram  again  it  will  be  evident  that  if  the  cations 
were  potassium  ions  the  loss  of  casein  from  the  anodal  region 
should  be  at  least  four  times  that  from  the  cathodal  region,  since 
the  equivalent  velocity  of  potassium  ions  is  at  least  four  times 
that  of  heavy  organic  ions.  The  experimental  fact  that  the  loss 
from  the  anodal  region  is  only  about  twice  as  great  as  that  from 
the  cathodal  region  can  only  be  interpreted  by  assuming  that 
protein  material  is  transported  into  the  cathodal  region  by  the 
current,  in  other  words,  that  the  current  is  transported  in  both  direc- 
tions by  protein  ions. 

4.  The  Migration- Velocity  of  Protein  Ions.  —  W.  B.  Hardy 
endeavored  to  measure  the  migration-velocities  of  serum-globu- 
lin ions  directly  (10)  by  placing  solutions  of  serum-globulin, 
combined  with  acids  or  bases,  at  the  bottom  of  a  U-tube  and 


188  ELECTROCHEMISTRY 

placing  above  the  solutions,  in  both  arms  of  the  U-tube,  a  solu- 
tion of  the  acid  or  base  employed  to  dissolve  the  protein.  A 
potential  gradient  was  placed  across  the  U-tube  and  it  was  ob- 
served that  in  acid  solutions  both  boundaries  of  the  protein  solu- 
tion migrated  towards  the  cathode,  while  in  alkaline  solutions 
both  boundaries  of  the  protein  solution  migrated  towards  the 
anode.  The  rate  of  migration  varied  between  7  to  10  X  10~5  cm. 
per  sec.  for  unit  potential  gradient  when  strong  acids  or  bases 
(HC1  and  NaOH)  were  employed  and  about  20  X  10~5  cm.  per 
sec.  when  a  weak  acid  (acetic  acid)  was  employed.  Following 
the  view  which  I  have  developed  of  the  mode  of  dissociation  of 
protein  salts,  it  would  appear  as  if  the  boundaries  should  move 
in  opposite  directions  at  approximately  equal  velocities.  A 
moment's  consideration  of  the  method  of  measurement  employed 
by  Hardy  shows,  however,  that  under  the  conditions  of  his  ex- 
periment this  would  not  occur.  At  the  boundary  of  the  protein 
solution  (nearly  neutral)  and  the  HC1  solution  (for  example)  a 
difference  of  potential  of  considerable  magnitude  would  exist 
owing  to  the  much  more  rapid  diffusion  of  H+  into  the  protein 
solution  than  of  Cl'.  This  difference  of  potential  would  lower  the 
potential  gradient  at  the  cathodal  boundary  and  raise  it  at  the 
anodal  boundary.  Bearing  in  mind  the  fact  that  the  two  protein 
ions  are  possessed  of  the  same  equivalent  migration-velocities 
under  unit  potential  fall,  the  effect  of  these  inequalities  in  poten- 
tial gradient  must  have  been,  at  the  anodal  boundary,  to  urge 
the  protein  anions  toward  the  anode  more  rapidly  than  the  cat- 
ions were  repelled  from  the  boundary,  and,  at  the  cathodal  bound- 
ary, to  repel  the  protein  anions  from  the  boundary  into  the  protein 
solution  more  rapidly  than  the  cations  crossed  the  boundary. 
The  net  result  of  these  processes  would  be,  obviously,  a  migration 
of  the  protein,  as  a  whole,  towards  the  anode.  The  contact- 
differences  of  potential  at  the  boundaries  would  be  equal  in 
magnitude  but  opposite  in  sense  and  hence  both  boundaries 
would  migrate  at  the  same  velocity,  but  in  the  same  direction. 
The  fact  that -the  highest  velocities  were  obtained  when  weak 
acids  (acetic)  were  employed,  in  which  the  difference  between 
the  equivalent  velocities- of  the  ions  (H+  and  acetanion)  is  great- 
est, strongly  supports  this  interpretation  of  Hardy's  results. 
Alkali-globulin,  in  contact  with  alkaline  solutions  would,  of 
course,  move  as  a  whole,  but  somewhat  more  slowly  (since  the 


THEORY  OF  PROTEIN  IONIZATION  189 

difference  between  the  velocity  of  OH7  and  that  of  Na+  is  not 
so  great  as  that  between  H+  and  Cl')  towards  the  cathode.  This 
'accords  with  the  experimental  observations  of  Hardy.  The 
velocities  measured  by  Hardy,  therefore,  do  not  afford  a  meas- 
ure of  the  migration-velocities  of  protein  ions  under  a  uniform 
potential  gradient. 

5.  Objections  to  the  above  Theory  of  Protein  lonization.  — 
Several  objections  to  the  hypothesis  of  protein  ionization  which 
has  been  presented  above  have  been  urged  by  Pauli,  Samec  and 
Strauss  (22).  These  objections  may  be  summarized  as  follows: 

(i)  The  hypothesis  involves  the  splitting  in  solution  of  a  poly- 
amino-acid  into  parts  which  are  not  amino-acids.  Enzymatic 
splitting  of  a  protein,  if  it  took  place  in  the  same  way,  would  not 
yield  amino-acid  end-products.  This  objection  is  really  analo- 
gous to  that  which  was  originally  advanced  against  Arrhenius' 
theory  of  the  electrolytic  dissociation  of  inorganic  substances. 
It  will  be  remembered  that  at  the  time  that  that  hypothesis  was 
first  advanced,  it  was  argued  that  the  ionization  of  KC1  into 
K+  and  Cl'  would  imply  the  presence  of  free  potassium  and  chlo- 
rine in  the  solution,  whereas  the  characteristic  properties  of  these 
substances  are  not  displayed  by  a  solution  of  KC1.  The  answer 
to  this  was  simply  that  the  properties  of  an  ion  are  not  to  be 
estimated  in  terms  of  those  of  a  molecule  and  furthermore  the 
ability  of  ions  in  a  solution  to  display  properties  independent  of 
those  of  the  oppositely  charged  ion  is  limited  by  electrostatic 
forces.  We  may  well  suppose  that  protein  ions,  like  other  ions 
in  solution,  cannot  react  with  other  substances  independently 
of  the  corresponding  ion  of  opposite  charge.  In  considering  the 
reaction  between  proteins  and  water  which  leads  to  the  decompo- 
sition of  the  polyamino-acid  chains  and  is  accelerated  by  enzymes 
we  must  consider  the  two  ions  of  the  protein  molecule  together, 
and  not  each  ion  separately.  Undoubtedly  the  hydrolytic  de- 
composition of  a  protein  must  involve  the  interaction  of  the 
protein  ions  with  accompanying  transfer  of  a  labile  hydrogen 
atom  from  the  cation  to  the  anion  in  accordance  with  the  scheme : 

H 

I 

-C  =  N-  +  HO.H=  -C-OH  +  N- 

I  II  I 

OH  OH 


1 90  ELECTROCHEMISTRY 

The  presence  of  a  labile  atom  in  a  molecule  leads,  not  unusually, 
to  the  development  of  color  in  solution,  due  to  the  absorption 
of  light-vibrations  by  the  vibrating  atom.  The  development  or 
non-development  of  absorption-bands  in  the  visible  or  photo- 
graphic spectrum  will,  of  course,  depend  upon  the  ratio  of  the 
frequency  of  vibration  of  the  labile  atom  to  the  frequency  of  the 
light-waves  which  impinge  upon  it.  That  the  presence  of  ab- 
sorption-bands in  the  visible  spectrum  is  not  an  inevitable  con- 
sequence of  the  presence  within  the  molecule  of  a  labile  hydro- 
gen atom  is  shown  by  the  analogous  instance  of  an  equilibrium 
between  the  keto-  and  enol-forms  in  the  colorless  hydantoins  (5). 

(ii)  The  above-mentioned  authors  still  consider  that  electro- 
phoresis  experiments  show  the  presence  of  only  one  protein  ion. 
Now  this  is  unquestionably  the  reverse  of  the  fact.  While  it  is 
true  that  a  superficial  consideration  of  Hardy's  results,  alluded  to 
in  the  preceding  section  of  this  chapter,  might  encourage  such  a 
supposition,  we  have  seen  that  more  careful  analysis  of  the  condi- 
tions of  the  experiment  shows  that  his  results  admit  of  a  very 
different  interpretation,  while  the  experiment  cited  at  the  end  of 
section  3  showing  that  in  solutions  of  casein  in  which  the  minimal 
valency  of  the  casein  ion,  on  the  supposition  that  potassium  casein- 
ate  yields  only  one  protein  ion,  must  be  4,  the  loss  of  protein  in 
the  cathodal  arm  during  electrolysis  is  only  one-half  the  loss  in 
the  anodal  arm,  is  totally  inconsistent  with  the  view  that  protein, 
during  electrolysis,  migrates  in  only  one  direction. 

Direct  proof,  however,  of  the  simultaneous  migration  of  protein 
to  both  poles  under  the  influence  of  an  electric  current  is  fortu- 
nately available.  Stirling  and  Brito  (35)  have  shown  that  if  a 
direct  current  be  allowed  to  traverse  a  solution  of  haemoglobin, 
deposition  of  crystals  of  haemoglobin  occurs  at  both  electrodes. 
An  alternating  current  is  without  effect.  Howell  (11)  has  fur- 
thermore shown  that  if  a  direct  current  be  passed  through  a 
solution  of  fibrinogen,  the  fibrinogen  increases  in  concentration 
at  both  poles  although,  as  might  be  anticipated,  the  solutions  of 
fibrinogen  obtained  from  the  neighborhood  of  the  two  poles  differ 
in  some  particulars  from  one  another  in  their  behavior  towards 
thrombin.  Since  haemoglobin  is  a  crystalline  protein  and  fibrin- 
ogen, as  Howell  has  recently  shown,  is  converted  by  thrombin 
into  a  crystalline  protein  (fibrin),  it  cannot  be  urged  that  in  these 
cases  we  are  dealing  with  the  opposite  migrations  of  two  distinct 


THEORY  OF  PROTEIN  IONIZATION  191 

and  oppositely  charged  colloids.  The  phenomena  can  only  be 
interpreted  to  mean  that  solutions  of  these  proteins  contain  oppo- 
sitely charged  but  otherwise  similar  moieties,  i.e.,  oppositely 
charged  protein  ions. 

(iii)  Pauli,  Samec  and  Strauss  also  urge  the  objection  that 
under  certain  conditions  (a  low  proportion  of  acid  to  protein) 
H+  and  Cl'  may  not  be  bound  equally  in  solutions  of  protein  in 
hydrochloric  acid.  This,  however,  does  not  really  affect  the 
question  of  the  mode  of  ionization  of  the  protein  molecule  but 
rather  the  question  of  the  relative  affinity  of  the  nitrogen  atom 
for  H+  and  Cl'  ions.  It  is  not  inconceivable  that  some  measure 
of  "Z witter  ion"  or  doubly  and  oppositely  charged  ion  formation 
may  occur  in  protein  solutions  (37).  Yet  this  is  not  the  normal, 
but  rather  the  exceptional  form  of  ionization  and  it  is  especially 
remarkable,  and  quite  inconsistent  with  the  view  that  proteins 
normally  dissociate  into  protein  and  non-protein  ions,  that  the 
inferiority  of  Cl'  binding  to  H+  binding  is  dependent  solely  upon 
the  proportion  of  HC1  to  protein  and  not  at  all  upon  the  dilution 
of  the  system,  although  the  equivalent  conductivity  of  protein 
solutions  is  very  decidedly  influenced  by  dilution  (Cf.  section  1). 

Perhaps  the  most  obvious  objection  to  the  hypothesis  which 
I  have  advanced  is  that  a  form  of  ionization  involving  the  break- 
ing of  a  double  bond  between  carbon  and  nitrogen  is  very  unusual 
and  in  fact  without  precedent  in  other  fields  of  chemistry.  It 
must  be  remembered,  however,  that  as  the  experiments  of  Gom- 
berg  (7)  have  so  beautifully  and  decisively  shown,  the  precise 
point  within  a  molecule  at  which  ionization  may  occur  is  de- 
termined by  the  strains  to  which  the  molecule  is  subjected,  and 
that  when  the  strain  is  unusually  great  the  break  involved  in 
ionization  may  occur  at  points  which  resist  the  tension  due  to 
strains  of  normal  magnitude.  Thus  ethane,  H3C  — CH3,  is  a  sub- 
stance of  remarkable  stability,  and  not  only  ethane,  but  tetra- 
phenyl  ethane,  (C6H5)2HC  —  CH(C6H5)2,  and  pentaphenyl  ethane 
(CeH5)  3C  —  CH(C6H5)2,  are  stable  substances.  Yet  all  attempts  to 
prepare  hexaphenylethane  have  failed  for  the  reason  that  the 
compound  breaks  in  the  middle,  the  bond  between  the  carbons, 
which  we  are  accustomed  to  regard  as  one  of  the  most  stable 
types  of  union,  being  ruptured  by  the  strain  imposed  upon  it  by 
the  great  weight  of  three  phenyl  groups  at  either  end  of  the 
molecule.  The  rupture  of  this  bond  is  all  the  more  remarkable 


1 92  ELECTROCHEMISTRY 

because  it  involves  the  creation  of  a  trivalent  carbon  atom  and 
the  independent  existence  of  an  unsaturated  radical.  Free  radi- 
cals containing  trivalent  carbon  have  been  prepared  from  com- 
pounds belonging  to  a  wide  variety  of  types,  and,  more  recently 
the  existence  of  divalent  nitrogen  has  also  been  recognized. 
With  facts  such  as  these  in  mind  the  customary  stability  of  any 
linkage  in  the  simpler  types  of  organic  compounds  no  longer 
suffices  as  a  priori  evidence  of  the  impossibility  of  its  rupture  in 
compounds  of  such  a  type  as  to  expose  it  to  strains  of  very  un- 
usual magnitude.  Now  the  protein  molecule  weighing  no  less 
than  from  10,000  to  20,000  and  consisting  of  a  large  number  of 
amino-acids  linked  together  in  long  chains  must  evidently  be 
subject  to  exceptional  strains.  Just  as  a  long  thin  bar  of  ma- 
terial, no  matter  how  great  its  strength,  will,  ultimately,  if  suffi- 
ciently long  and  unsupported,  break  of  its  own  weight,  so  in  the 
long  chain  of  atoms  composing  a  protein  molecule,  rupture  of 
linkages  may  occur  which  are  sufficiently  strong  to  resist  all  the 
strains  to  which  shorter  or  more  symmetrical  molecules  are  com- 
monly subjected.  It  may  of  course  be  urged  that  this  argument 
proves  too  much,  since,  by  parity  of  reasoning,  the  split  of  the 
linkage  between  carbon  and  nitrogen  should  occur  at  all  times  and 
not  be  dependent  upon  salt-formation,  the  additional  strain  im- 
posed by  the  weight  of  a  molecule  of  hydrochloric  acid  or  sodium 
hydroxide  being  negligible  in  comparison  with  the  total  strain  to 
which  the  molecule  is  subjected.  It  must  be  remembered,  how- 
ever, that  the  additional  strains  which  a  molecule  of  acid  or  base 
introduces  into  the  molecule  are  not  merely  those  commensurate 
with  and  attributable  to  its  weight,  but  also  strains  of  electrostatic 
origin,  since  the  salt  which  is  formed  unquestionably  undergoes 
ionization.  It  may  very  possibly  be  true  that  the  first  step  in 
salt  formation  consists  in  the  neutralization  of  end  —  NH2  or 
—  COOH  groups,  but  that  the  ionization  of  the  compound  formed, 
leading  to  the  development  of  electrostatic  tension  at  the  very 
places  at  which  it  must  exert  the  greatest  strain,  namely  the  ex- 
tremities of  the  molecule,  results  in  the  splitting  of  the  otherwise 
stable  linkage  —  C  =  N  —  and  the  redistribution  of  the  compo- 

I 
nents  of  the  molecule  and  the  strains  to  which  it  is  subjected. 

6.   Biological  Applications;  the  * 'Selective"  Action  of  Living 
Tissues.  —  The  non-dissociable  character  of  the  inorganic  con- 


BIOLOGICAL  APPLICATIONS  193 

stituent  of  the  protein  salts  affords,  I  believe,  an  explanation  of 
an  important  physiological  phenomenon;  I  refer  to  the  well- 
known  power  which  living  tissues  possess  of  " selecting"  or  storing 
up  certain  inorganic  constituents  in  a  concentration  greater  than 
that  in  which  these  substances  are  found  in  the  surrounding 
liquid  medium  (23)  (25)  (26).  Thus  the  skeletal  muscles  and  the 
red  blood  corpuscles  contain  a  marked  excess  of  potassium  over 
sodium,  while  the  plasma  which  bathes  them  contains  much  less 
potassium  than  sodium.  Again,  although  in  fresh- water  streams 
the  concentration  of  potassium  salts  is  often  very  low,  the  plants 
which  live  in  them  are  capable  of  storing  up  a  comparatively 
large  amount  of  potassium  in  their  tissues. 

If  we  place  within  a  dialyser  an  excess  of  diffusible  potassium 
salts  over  diffusible  sodium  salts  and  dialyse  against  a  solution 
containing  excess  of  diffusible  sodium  salts,  the  proportions  of 
sodium  to  potassium  within  and  without  the  dialyser  sooner  or 
later  readjust  themselves,  approaching  equality.  Hence  the  above- 
mentioned  phenomenon,  which  is  met  with  in  living  tissues, 
admits,  as  Loeb  has  pointed  out  (14)  of  only  one  explanation,  — • 
the  inorganic  constituents  of  a  tissue  which  are  found  therein  in 
excess  of  their  concentration  in  the  fluids  which  bathe  it  must 
exist  within  the  tissue  in  the  form  of  non-dissociated  non-diffu- 
sible compounds.  "If  a  tissue  utilizes  one  kind  of  metal  in  this 
way,  for  example  K,  while  another  metal,  for  example  Na,  is 
chiefly  used  for  the  formation  of  dissociable  compounds  with 
Na  as  the  free  ion,  the  consequence  will  be  that  the  ashes  of  the 
tissue  contain  K  and  Na  in  altogether  different  proportions  from 
those  in  which  they  are  contained  in  the  surrounding  solution. 
I  think  we  may  take  it  for  granted  that,  at  least,  potassium  forms 
a  non-dissociable  constituent  of  the  protoplasm  of  a  number  of 
tissues  of  animals  and  plants"  (Loeb.  loc.  cit.). 

If  we  admit  that,  as  many  investigators  now  believe  (13)  (14) 
(32)  (26),  the  inorganic  constituents  of  living  tissues  are  partly 
united  with  proteins,  the  fact  that  such  unions  dissociate  only 
into  protein  and  not  into  protein  and  inorganic  ions  affords  a 
sufficient  explanation  of  the  above  phenomenon. 

Loeb  (13)  (14)  and  W.  A.  Osborne  (21)  have  advanced  an 
analogous  explanation  of  the  "  oligodynamic "  (16)  (19)  action  of 
many  highly  toxic  heavy  metals. 


194  ELECTROCHEMISTRY 


LITERATURE   CITED 

(1)  Beutner,  R.,  Biochem.  Zeit.  47  (1912),  p.  73. 

(2)  Blasel,  L.,  and  Matula,  J.,  Biochem.  Zeit.  58  (1914),  p.  417. 

(3)  Bredig,  G.,  Zeit.  f.  physik.  Chem.  13  (1894),  p.  191.  ""    ... _ 

(4)  Bugarszky,  S.,  and  Liebermann,  L.,  Arch,  f .  d.  Ges.  Physiol.  72  (1898), 

p.  51. 

(5)  Dakin,  H.  D.,  Journ.  Biol.  Chem.  13  (1912-13),  p.  357;  Amer.  Chem. 

Journ.  44  (1910),  p.  48. 

(6)  van  Dam,  W.  Zeit.  f .  physiol.  Chem.  58  (1908),  p.  295;  61  (1909),  p.  147. 

(7)  Gomberg,  M.,  Journ.  of  Indust.  &  Engineering  Chem.  6  (1914),  p.  33. 

(8)  Guthe,  K.  E.,  Bull.  U.  S.  Bureau  of  Standards  (1905),  p.  362. 

(9)  Hardy,  W.  B.,  Journ.  of  Physiol.  24  (1899),  p.  288. 

(10)  Hardy,  W.  B.,  Journ.  of  Physiol.  33  (1905),  p.  286. 

(11)  Howell,  W.  H.,  Amer.  Journ.  of  Physiol.  40  (1916),  p.  526. 

(12)  Kohlrausch,  F.,  and  Holborn,  L.,  "Das  Lietvermogen  der  Elektro- 

lyte,"  Leipzig,  1898. 

(13)  Loeb,  Jacques,  Amer.  Journ.  of  Physiol.  3  (1900),  p.  327. 

(14)  Loeb,  Jacques,  "The  Dynamics  of  Living  Matter,"  New  York  (1906). 

(15)  Loevenhart,  A.  S.,  Zeit.  f.  physiol.  Chem.  41  (1904),  p.  176. 

(16)  Loew,  O.,  Landw.  Jahrb.  20  (1891),  p.  235. 

(17)  Manabe,  K.,  and  Matula,  J.,  Biochem.  Zeit.  52  (1913),  p.  269. 

(18)  Moore,  P.,  Roaf,  H.  E.,  and  Webster,  A.,  Biochem.  Journ.  6  (1912), 

p.  110. 

(19)  Nageli,  O.,  Denkschr.  der  Schweiz.  Naturforsch.  Ges.  33  (1893),  p.  1. 

(20)  Oryng,  T.,  and  Pauli,  W.,  Biochem.  Zeit.  70  (1915),  p.  368. 

(21)  Osborne,  W.  A.,  Journ.  of  Physiol.  34  (1906),  p.  84. 

(22)  Pauli,  W.,  Samec,  M.,  and  Strauss,  E.,  Biochem.  Zeit.  59](1914),  p.  470. 

(23)  Robertson,  T.  Brailsford,  Journ.  of  physical  Chem.  11  (1907),  p.  542. 

(24)  Robertson,  T.  Brailsford,  Journ.  of  physical  Chem.  13  (1909),  p.  469. 

(25)  Robertson,  T.  Brailsford,  Univ.  of  Calif.  Publ.  Physiol.  3  (1909),  p. 

170. 

(26)  Robertson,  T.  Brailsford,  Ergeb.  d.  Physiol.  10  (1910),  p.  334. 

(27)  Robertson,  T.  Brailsford,  Journ.  of  physical  Chem.  14  (1910),  p.  528. 

(28)  Robertson,  T.  Brailsford,  Journ.  of  physical  Chem.  14  (1910),  p.  601. 

(29)  Robertson,  T.  Brailsford,  Journ.  of  physical  Chem.  15  (1911),  p.  179. 

(30)  Rohmann,  F.,  and  Hirschstein,  L.,  Beitr.  z.  chem.  Physiol.  und  Path. 

3  (1902),  p.  288. 

(31)  Rohonyi,  H.,  Biochem.  Zeit.  44  (1912),  p.  165. 

(32)  Richards,  T.  W.,  Journ.  of  physical  Chem.  4  (1900),  p.  207. 

(33)  Ringer,  W.  E.,  Zeit.  f.  physiol.  Chem.  95  (1915),  p.  195. 

(34)  Sackur,  O.,  Zeit.  f.  physik.  Chem.  41  (1902),  p.  672. 

(35)  Sjoquist,  J.,  Skand.  Arch.  f.  physiol.  5  (1895),  p.  277. 

(36)  Stirling  W.,  and  Brito,  P.  S.,  Journ.  Anat.  and  Physiol.  16  (1882),  p.  446. 

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CHAPTER  IX 
THE   COMBINING   CAPACITY   OF  THE  PROTEINS 

1.  The  Electrochemical  Determination  of  the  Combining 
Capacity  of  the  Proteins.  —  The  potentiometric  method  was 
employed  by  Bugarszky  and  Liebermann  (5)  for  the  purpose  of 
demonstrating  that  proteins  possess  a  true  combining  capacity 
for  acids  and  bases.  I  have  utilized  this  method  in  determining 
the  relationship  of  the  combining  capacity  of  the  proteins  to  the 
alkalinity  or  acidity  as  well  as  the  absolute  concentration  of  their 
solutions  (54)  (59)  (60).  In  brief,  the  principles  upon  which  the 
method  depends  are  as  follows:* 

When  a  metal  or  hydrogen  is  brought  into  contact  with  a  liquid 
a  certain  amount  of  the  metal  tends  to  pass  over  into  the  liquid 
and  it  strives  to  do  so  with  a  certain  measurable  and  character- 
istic force,  analogous  to  gas-  or  osmotic-pressure,  which  Nernst 
(47)  (48)  has  termed  the  solution  pressure.  The  particles  which 
this  pressure  tends  to  bring  into  solution  are  all,  in  the  case  of 
the  metals  and  hydrogen,  charged  electropositively.  The  number 
of  these  particles  which  actually  pass  into  solution,  provided  no 
current  is  allowed  to  traverse  the  liquid  and  the  metal,  and  no 
chemical  work  is  performed,  must  be  very  small  and,  in  proportion 
to  weighable  qualities,  quite  evanescent;  for  as  the  positively 
charged  particles  pass  out  into  the  liquid  an  electrostatic  tension 
is  necessarily  developed.  The  film  of  liquid  which  is  in  immediate 
contact  with  the  metal  becomes  positively  charged;  the  metal, 
having  lost  positive  charges,  acquires  a  corresponding  negative 
charge,  and  the  force  driving  fresh  metal  or  hydrogen  ions  into 
the  liquid  becomes  balanced  by  the  electrostatic  repulsion  of  the 
positive  ions,  from  the  film  of  liquid  which  is  in  contact  with  the 
metal  into  the  surface  of  the  metal  again.  Hence  there  is  quickly 
developed  a  certain  constant  difference  of  electric  potential  be- 

*  For  a  fuller  discussion  of  these  principles  and  of  the  forms  of  apparatus 
used  the  reader  is  referred  to  standard  works  of  general  physical  chemistry 
(21)  (41)  (64)  (67). 

195 


1 96  ELECTROCHEMISTRY 

tween  the  liquid  (charged  positively)  and  the  metal  (charged 
negatively)  which  is  characteristic  for  each  metal  and  each  liquid. 

The  force  which  tends  to  drive  the  metal  ions  back  into  the 
metal  again  is  the  osmotic  pressure  of  the  dissolved  ions.  If, 
through  the  introduction  of  a  dissociable  salt  of  the  metal,  the 
concentration  (osmotic  pressure)  of  the  metal  ions  in  the  liquid 
is  increased,  this  may  equal  or  even  exceed  the  solution  pressure 
of  the  metal  itself.  In  the  former  case  the  potential  difference 
between  the  metal  and  the  solution  is  abolished;  in  the  latter  it 
is  renewed  again,  but  in  the  opposite  sense,  the  metal  being  now 
charged  positively  and  the  solution  negatively. 

If,  now,  we  build  up  a  galvanic  element  as  follows:  Metallic 
silver  in  contact  with  dilute  AgN03,  in  conducting  communi- 
cation with  strong  AgN03,  the  latter  solution  being  again  in  con- 
tact with  metallic  silver,  it  is  evident  that  there  must  be  a  differ- 
ence in  potential  between  the  two  portions  of  metallic  silver,  a 
potential  which  will  be  greater  the  greater  the  difference  between 
the  concentrations  of  the  two  silver  nitrate  solutions.  Provided 
the  potential  between  the  two  silver  nitrate  solutions  themselves 
(due  to  the  unequal  migration-velocities  of  silver  and  N03  ions) 
can  be  neglected  or  made  to  vanish  (and  we  will  shortly  explain 
how  this  may  be  approximately  achieved),  it  has  been  shown 
by  Nernst  that  the  potential  of  this  " concentration  chain"  can 
be  expressed  by  the  formula: 

_  RT  C2 

It  —     77»    '  lOgnat.   s*i 

r  TI  (_/  i 

where  R  is  the  gas  constant,  T  the  absolute  temperature,  F  the 
Faraday  constant,  77  the  valency  of  the  ion  (in  this  case  1),  Cz  the 
concentration  of  the  stronger  solution  of  silver  nitrate,  Ci  that  of 
the  weaker  and  TT  the  potential  in  volts. 

In  this  way  the  absolute  concentrations  of  two  different  solu- 
tions of  an  ion  can  be  accurately  computed,  provided  only  a 
minute  amount  of  current  be  allowed  to  pass^  during  the  potential 
determination,  without  any  significant  alteration  in  the  compo- 
sition of  the  solutions.  The  measurement  is,  in  other  words, 
static;  and  static  methods  of  measurement  must  be  employed  in 
determining  the  combining  capacities  of  proteins  because  the 
combining  capacity  of  proteins  is  not  possessed  of  one  fixed  value 
but  of  fixed  minimum  and  maximum  values  and,  between  these 


COMBINING  CAPACITY  197 

intermediate  values  which  are  determined  by  the  alkalinity  or 
acidity  (H+  concentration)  of  their  solutions. 

In  applying  this  method  to  the  comparison  of  the  acidity  or 
alkalinity  (H+  concentration)  of  solutions  the  terminal  electrodes 
must  be  hydrogen.  A  hydrogen  electrode  can  be  obtained  by 
employing  a  saturated  solution  of  hydrogen  in  a  metal,  e.g., 
platinum.  In  order  to  obtain  a  large  surface,  platinum  gauze 
coated  with  platinum  black  is  usually  employed  and  a  stream 
of  pure  hydrogen  is  passed  over  or  through  the  gauze.  The 
potential  between  the  two  acid  or  alkaline  solutions  themselves 
(as  distinguished  from  that  between  the  hydrogen  electrodes  and 
these  solutions)  may  be  abolished  or  at  any  rate  greatly  diminished 
by  connecting  them  by  means  of  a  U-tube  filled  with  agar  satu- 
rated with  KC1  (3)  (1). 

I  have  utilized  this  method  in  determining  the  combining 
capacities  of  casein  and  of  ovomucoid  for  acids  or  bases  in  solu- 
tions of  varying  acidity  and  alkalinity  (59)  (60).  For  further 
details  regarding  the  technique  employed  and  the  precautions  to 
be  taken  in  carrying  out  such  determinations  in  solutions  which 
contain  proteins,  the  reader  is  referred  to  the  appendix. 

In  the  following  tables,  which  enumerate  the  results  of  these 
experiments,  the  symbols  which  are  employed  have  the  following 
significance : 

61  ==  The  concentration  of  a  solution  of  base  in  which  protein 

is  dissolved, 
ai  =  The  concentration  of  a  solution  of  acid  in  which  protein 

is  dissolved. 
TT  =  The  potential  of  the  chain : 


acid  or  base 
plus  protein 


acid  or  base 
alone 


in  volts.     At  30  degrees,  with  monacid  bases  or  mono- 
basic acids 

=  0.0601  loglo  j    or  0.0601  loglo  ~ 

b  =  The  hydroxyl  concentration  (unneutralized  base)  in  the 

solution  of  base  containing  protein. 
a  =  The  hydrogen  concentration  (unneutralized  acid)  in  the 

solution  of  acid  containing  protein. 
m  =  bi  —  b  or  a\  —  a  =  the  concentration  of  base  or  acid 

neutralized  by  the  protein. 


198 


ELECTROCHEMISTRY 


To  all  the  solutions  employed  a  small  amount  of  KC1  was  added. 
This  is  necessitated  in  the  preparation  of  the  casein  solutions 
by  technical  difficulties  of  which  an  account  will  be  found  in  the 
appendix,  but  it  serves  also  to  confer  upon  the  solutions  a  higher 
conductivity  and  thus  to  render  more  easy  the  detection  of  the 
zero  point  upon  the  potentiometer  bridge  wire  by  means  of  a 
galvanometer.  So  far  as  the  solutions  of  casein  in  KOH  are 
concerned,  the  presence  of  KC1  in  the  concentrations  employed 
evidently  did  not  appreciably  affect  the  combining  capacity  of 
the  protein  for  the  base,  since,  as  the  tables  reveal,  in  solutions 
of  very  different  KCl-content  (0.015  N  to  0.030  N)  the  combining 
capacity  at  absolute  neutrality  proved  to  be  the  same.  In  the 
tables  the  total  amount  of  potassium  present  in  the  solvent  as 
KOH  or  KC1,  is  designated  by  the  symbol  r. 

All  of  these  determinations  were  made  at  30°  C. 


0.5  PER  CENT  CASEIN  DISSOLVED  IN  KOH  SOLUTIONS 

(r  =  0.020) 


&1 

it 

b 

m 

0.02000 

0.0160 

1.083X10-2 

0.00917 

0.01000 

0.0462 

1.701X10-3 

0.00830 

0.00750 

0.0708 

4.987X10-4 

0.00700 

0.00500 

0.1181 

5.429X10"5 

0.00495 

0.00250 

0.2600 

1.181X10-7 

0.00250 

0.00150 

0.2710 

4.648X10-8 

0.00150 

1.0  PER  CENT  CASEIN  IN  KOH  SOLUTIONS  (FIRST  SERIES) 


bi 

7T 

b 

m 

r 

Remarks 

0.03028 

0.0239 

1.213X10-2 

0.01815 

0.03028 

0.02907 

0.0273 

1.021X10-2 

0.01886 

0.03028 

0.02422 

0.0320 

7.111X10-3 

0.01711 

0.02422 

0.01817 

0.0538 

2.314X10-3 

0.01586 

0.01817 

0.01212 

0.0937 

3.351X10-4 

0.01178 

0.01817 

0.01091 

0.1042 

2.015X10-4 

0.01071 

0.01817 

0.00970 

0.1332 

5.907X10-5 

0.00964 

0.01817 

0.00849 

0.1736 

1.096X10-5 

0.00848 

0.01817 

0.00728 

0.2276 

1.191X10-6 

0.00728 

0.01817 

0.00607 

0.2542 

3.583X10-7 

0.00607 

0.01817 

0.00486 

0.2758 

1.251X10-7 

0.00486 

0.01817  ) 

Av.  value  of 

0.00486 

0.2753 

1.276X10-7 

0.00486 

0.01817  f 

7r  =  0.2756 

0.00365 

0.3034 

3.265X10-8 

0.00365 

0.01817 

0.00244 

0.3130 

1.514X10~8 

0.00244 

0.01817 

COMBINING  CAPACITY 


199 


1.0  PER  CENT  CASEIN  KOH  SOLUTIONS    (SECOND  SERIES) 

(r  =  0.030) 


6) 

•K 

6 

TO 

0.03000 

0  0231 

1.237X10-2 

0.01763 

0.02500 

0  0313 

7.523X10- 

0.01748 

0.02000 

0  0471 

3.292X10- 

0.01671 

0.01750 

0  0603 

1.736X10- 

0.01576 

0  01500 

0.0741 

8.782X10- 

0.01412 

0.01250 

0  0954 

3.233X1  - 

0.01218 

0.01000 

0.1299 

6.892X10- 

0.00993 

0  00750 

0.2265 

1.276X10- 

0.00750 

0  00500 

0.2596 

2.398X10- 

0.00500 

0  00250 

0  2903 

3.699X10- 

0.00250 

1.5   PER  CENT   CASEIN   IN   KOH  SOLUTIONS    (r  =  0.030) 


61 

•a 

b 

TO 

0  03000 
0  02000 
0  01500 
0.01000 
0.00750 
0.00500 

0.0439 
0.0881 
0.1280 
0  2523 
0.2897 
0.3128 

5.578X10-3 
6.842X10-4 
1.  113X10-' 
6.337X10-7 
1.136X10-7 
3.  119X10-8 

0.02442 
0.01932 
0.01489 
0.01000 
0.00750 
0.00500 

2.0  PER  CENT  CASEIN  IN  KOH  SOLUTIONS 


6, 

TT 

6 

TO 

r 

0.05000 
0.03000 
0.02000 
0.01500 
0.01000 
0  00500 

0.0311 
0.0778 
0.1343 
0.2425 
0.3000 
0.3152 

1.522X10-2 
1.525X10-3 
1.167X10-4 
1.  383X10-' 
1.020X10-7 
2.846X10-8 

0.03478 
0.02847 
0.01988 
0.01500 
0.01000 
0.00500 

0.050 
0.030 
0.040 
0.040 
0.040 
0.040 

3.0  PER  CENT  CASEIN  IN  KOH  SOLUTIONS 


&» 

ir 

b 

TO 

r 

o.osooo 

0.0733 

3.011X10-3 

0.04699 

0.050) 

Av.value  of 

0.05000 

0.0739 

2.943X10'3 

0.04706 

o.osot 

m=  0.04703 

0.03000 

0.1438 

1.217X10'4 

0.02988 

0.040 

0.02500 

0.2343 

3.156X10-6 

0.02500 

0.040 

0.02000 

0.2823 

4.017X10-7 

0.02000 

0.040 

0  01500 

0.3172 

7.920X10'8 

0.01500 

0.040 

0  01000 

0.3413 

2.096X10-8 

0.01000 

0.040 

200 


ELECTROCHEMISTRY 


The  relation  between  m,  the  amount  of  alkali  neutralized  by  the 
casein,  and  61,  the  alkalinity  of  the  solution  in  which  the  casein 
was  dissolved,  is  shown  graphically,  for  all  of  the  concentrations 
of  casein  employed,  in  the  accompanying  figure : 

0.05 


0.04 


0.03 


0.02 


0.01 


b, 


0.01 


0.02 


0.03 


0.04 


0.05 


The  ordinates  =  m  =  the  concentration  of  KOH  neutralized 

by  0.5%,  1%,  1.5%,  2.0%  and  3%  casein. 

The  abscissae  =  6:  =  the  concentration  of  the  KOH  solution 

in  which  the  casein  was  dissolved. 

It  will  be  understood,  of  course,  that  the  curves  only  represent 
this  relation  for  the  alkalinities  of  the  original  solutions  in  excess 
of  that  necessary  to  dissolve  all  of  the  casein.  For  an  ordinary 
acid,  forming  only  one  salt  with  the  base,  which  did  not  undergo 
hydrolytic  dissociation,  the  curve  would,  of  course,  be  a  straight 
line  parallel  with  the  horizontal  axis.  It  will  be  seen  that  as  the 
proportion  of  base  to  casein  declines,  the  combining  capacity  of 
the  casein  tends  to  become  directly  proportional  to  the  concen- 
tration of  the  base,  but  that  as  the  proportion  of  base  to  casein 
(and  the  excess  of  unneutralized  base)  becomes  large,  the  com- 
bining capacity  of  the  casein  tends  towards  constancy,  i.e.,  in 
comparatively  strongly  alkaline  solutions  the  behavior  of  casein 
approximates  more  and  more  to  that  of  an  ordinary  acid. 

These  results  are  to  be  interpreted  as  follows:  When  just 
enough  alkali  (=  11.4  X  10~5  equivalents  per  gram,  Cf.  Chap.  V) 


COMBINING  CAPACITY  201 

has  reacted  with  casein  to  dissolve  it,  we  may  suppose  that  one 

—  N.HOC—  group  (or  two  such  groups,  if  dicarboxylic  radicals 
are  involved)  in  each  molecule  of  casein  has  been  opened  up, 
in  accordance  with  the  equation:  jj 

^  '       ++ 
-N.HOC-  +  KOH  =  -  ^N  +  KOC- 

I 
OH 

From  the  Guldberg  and  Waage  mass  law  we  know  that  if  this 
equation  represents  the  true  nature  of  the  reaction  which  occurs, 
then  the  mass  of  caseinate  formed  must  be  dependent  (the  tem- 
perature and  other  conditions  of  the  reaction  being  constant) 
only  upon  the  reacting  masses  of  free  casein  and  KOH  and  not 
upon  the  total  dilution  of  the  system.  At  the  completion  of  the 
reaction  these  masses  are  both  very  small,  since  free  casein  is 
inappreciably  soluble  in  water  and  the  solution  of  this  salt  of 
casein  is  acid  in  reaction  (Cf.  Chap.  V).  Upon  further  addition 
of  alkali,  however,  another  —N.HOC—  group  (or  pair  of  groups) 
is  opened  up  and  when  another  11.4  X  10~5  equivalents  of  alkali 
have  been  added  per  gram  of  casein  we  have  now  four  ions  of 
caseinate  instead  of  two.  Whether  these  ions  are  derived  from 
one,  or  from  two,  molecules  of  caseinate  these  data  do  not  enable 
us  to  decide.  As  the  tables  show,  the  solution  of  this  salt  is  also 
acid  to  litmus.  Upon  still  further  addition  of  alkali  yet  another 

—  N.HOC—   group  (or  pair  of  groups)  in  each  casein  molecule 
is  opened  up,  and  when  22.8  X  10~5  more  equivalents  of  KOH 
(=  45.6  X  10~5  per  gram  in  all)  have  been  added  all  of  the  ions 
of  caseinate  have  again  been  split  in  half.     The  solution  of  this 
salt  is,  as  we  shall  see,  almost  exactly  neutral  to  litmus.     Again 
doubling  the  alkali-content  the  ions  will  be  split  again,  yielding 
a  salt  containing  91.2  X  10~5  equivalents  of  KOH  per  gram,  the 
solution  of  which,  as  the  above  tables  reveal,  is  alkaline  to  phenol- 
phthalein   (62)   and  which,  therefore,  presumably,  requires  the 
presence  of  a  slight  excess  of  KOH  to  maintain  its  stability,  i.e., 
to  push  the  equilibrium  in  the  above  reaction  sufficiently  over 
to  the  right.    We  may  suppose  that  this  type  of  reaction  is  re- 
peated until  the  last  —N.HOC—  group  that  is  dissociable  by 
alkali  has  been  opened  up,  when  the  combining  capacity  of  the 
casein  reaches,  as  we  have  seen,  a  constant  maximal  value.     This 
maximum  value  of  the  combining  capacity  must  necessarily,  if 


202 


ELECTROCHEMISTRY 


the  above  picture  of  the  chain  of  events  is  correct,  be  an  even 
multiple  (1,  2,  4,  8,  16,  etc.)  of  the  quantity  of  alkali  which  is 
just  sufficient  to  form  the  first  salt,  i.e.,  to  carry  the  casein  into 
solution.  By  interpolation  from  the  above  tables  and  by  con- 
tinuing the  curves  it  is  seen  that  for  all  of  the  percentages  of  casein 
employed  the  constant  maximal  value  of  the  combining  capacity 
for  KOH  is,  as  nearly  as  can  be  estimated,  180  X  10~5  equiva- 

1 80 

lents  per  gram.  Now  -^r-r  =  15.8  or,  within  the  error  of  esti- 
mation, 16.  The  experimental  results  are  therefore  in  good 
agreement  with  the  theory  and  we  may  conclude  that  casein 
forms  the  following  salts  with  KOH: 

I  containing    11.4  X  10~6  equivalents  of  KOH  per  gram  of  casein 
II         "  22.8  X  10-5 

III  "  45.6X10-6  "  "  "  " 

IV  "  91.2X10-5  "  "  "  " 
V         "          182.4  X  ID"6           "                  "               "  " 

In  forming  the  last  of  these  salts  16  —  N.HOC—  groups  (or  a 
multiple  of  this  number)  have  been  opened  up. 

Similar  experiments  were  carried  out  upon  solutions  of  ovo- 
mucoid  in  dilute  acid  and  alkali;  they  are  tabulated  below: 

1  PER  CENT  OVOMUCOID  IN  KOH  SOLUTIONS 
(KC1  in  each  solution  =  0.01  N) 


61 

IT 

b 

m 

0.0005 

0.0755 

2.77X10-5 

0.00047 

0.0010 

0.0475 

1.62X10-4 

0.00084 

0.0020 

0.0500 

2.95X10-4 

0.00171 

0.0030 

0.0367 

7.34X10-4 

0.00227 

0.0050 

0.0356 

1.28X10-3 

0.00372 

0.0100 

0.0185 

4.92X10-3 

0.00508 

1  PER  CENT  OVOMUCOID  IN  HCL  SOLUTIONS 
(KC1  in  each  solution  =  0.01  N) 


fll 

tc 

a 

m 

6.95XlO-« 

0.1087 

1.08X10-7 

6.84X10-6 

0.0005 

0.2261 

8.65X10-8 

0.00050 

0.0010 

0.2107 

3.12X10-7 

.  0.00100 

0.0020 

0.17Z6 

2.69X10-6 

0.00198 

0.0030 

0.1492 

9.88X10-* 

0.00299 

0.0050 

0.1190 

5.24X10-5 

0.00495 

0.0100 

0.0610 

9.66X10-4 

0-00903 

COMBINING  CAPACITY  203 

The  results  are  shown  graphically  in  the  accompanying  figure: 


0.01 


0.005 


b,  . 


0.01 


0.005 


0.01 


0.005 


61  is  the  concentration  of  KOH  in 
which  1  per  cent  ovomucoid  is  dis- 
solved; m  is  the  number  of  gram- 
equivalents  of  KOH  which  is  neutral- 
ized per  litre. 


0.006  0.01 

«i  is  the  concentration  of  HC1  in 
which  1  per  cent  ovomucoid  is  dis- 
solved; m  is  the  number  of  gram- 
equivalents  of  HC1  which  is  neutral- 
ized per  litre. 


It  is  obvious  that  the  relationship  between  the  amount  of 
acid  or  base  neutralized  by  the  ovomucoid  and  the  total  amount 
of  base  present  is  of  very  much  the  same  general  nature  as  the 
corresponding  relationship  for  solutions  of  casein  in  KOH  — 
solutions.  Ovomucoid,  however,  differs  very  strikingly  from 
casein  in  the  fact  that  it  is  predominantly  basic,  while  casein  is 
predominantly  acid.  The  maximum  combining  capacity  of  ovo- 
mucoid for  KOH  is  obviously  about  50  X  10~5  equivalents  per 
gram,  but  its  maximum  combining  capacity  for  acids  was  not 
attained  in  any  of  the  solutions  investigated  and  must  be  at  least 
100  X  10~5  equivalents  per  gram.  Since  free  ovomucoid,  unlike 
free  casein,  is  soluble  in  water  the  quantity  of  a  base  or  acid  which 
enters  into  one  —  N.HOC—  group  cannot  be  estimated  from 
these  data  alone,  but  it  is  evident  that  it  cannot  be  in  excess  of 
50  X  10~5  per  gram  of  protein. 

The  combining  capacities  of  serum  albumin,  gelatin  and  de- 
aminized  gelatin  for  acids  have  been  determined  electrometri- 
cally  by  Pauli  and  Hirschfeld  (49).  They  find  that,  as  in  the 
case  of  casein,  the  maximum  combining  capacities  of  these  pro- 
teins are  independent  of  their  dilution  and,  moreover,  that  in 
isohydric  solutions  acetic  acid,  despite  the  fact  that  it  yields  a 
much  inferior  proportion  of  hydrogen  ions,  is  bound  by  proteins 
in  greater  proportion  than  hydrochloric  acid,  a  fact  which  in 


204  ELECTROCHEMISTRY 

itself  speaks  very  strongly  in  favor  of  the  view  that  the  protein 
compounds  which  are  formed  do  not  yield,  by  electrolytic  disso- 
ciation, the  corresponding  acid  anions.  They,  moreover,  find 
that  the  combining  capacity  of  deaminized  gelatin  is  hardly 
inferior  to  that  of  normal  gelatin. 

The  electrometric  method  of  measuring  the  combining  capaci- 
ties of  proteins  for  acids  and  bases  has  also  been  employed  by 
D'Agostino  and  Quagliariello  (2),  Rohonyi  (61),  Manabe  and 
Matula  (38),  Blasel  and  Matula  (4),  Ringer  (52)  and  Schmidt 
(63)  (64). 

2.  The  Combining  Capacity  of  Casein  for  Bases  and  of  Ovo- 
mucoid  for  HC1  at  Absolute  Neutrality.  —  A  number  of  investi- 
gators have  shown  (66)  (34)  (71),  by  direct  titration  to  neutrality 
to  litmus,  that  the  combining  capacity  of  casein  at  absolute 
neutrality  is  constant,  that  is,  independent  of  the  total  dilu- 
tion of  the  system.  The  potentiometric  determinations,  cited  in 
the  above  tables,  enable  us  to  confirm  these  observations.  It 
will  be  recollected  that  the  OH'  concentration  in  the  solutions 
containing  casein  was  determined  by  measuring  the  potential 
between  two  hydrogen  electrodes,  the  one  dipped  in  a  solution 
containing  a  given  concentration  of  casein,  the  other  in  an  ex- 
actly similar  solution  to  which  no  casein  had  been  added.  Plot- 
ting a  curve  in  which  the  reaction  of  the  solutions  containing 
no  casein  form  the  abscissae  and  the  potentials  between  the  two 
solutions  the  ordinates,  the  reactions  ( =  x)  of  the  solution  to  which 
the  given  concentration  of  casein  had  to  be  added  in  order  to 
procure  an  exactly  neutral  solution  *  is  given  by  the  intersection 
of  this  curve  with  the  curve  defined  by  the  formula: 

y  =  0.4107  -  0.0601  Iog10  x. 

The  points  of  intersection  of  these  curves  may  be  found,  with 
sufficient  approach  to  accuracy,  in  the  following  way:  The  values 
of  y  in  the  above  curve  corresponding  to  x  (alkalinity  of  the 
solution  containing  no  casein)  =  0.0025;  0.005;  0.0075;  0.010; 
0.015  and  0.020  are  computed  and  these  points  are  marked  upon 
accurately  ruled  "squared  paper"  and  joined  by  straight  lines. 
Experimental  values  of  TT-  lying  upon  each  side  of  its  value  at 
neutrality,  in  each  solution,  are  then  also  marked  off  upon  the 

*  Taking  the  H+  concentration  at  absolute  neutrality  at  30  degrees  as 
1.47  X  10-*;  cf.  Kohlrauch  and  Heydweiller  (33). 


CASEIN  AND  OVOMUCOID 


205 


paper  and  joined  by  straight  lines.  The  abscissa  of  the  point  of 
intersection  of  the  two  broken  curves  thus  drawn  yields  the  number 
of  gram  equivalents  of  KOH  which  are  bound  by  the  given  per- 
centage of  casein  in  the  production  of  an  absolutely  neutral 
solution.*  Dividing  the  number  of  gram  equivalents  of  KOH 
neutralized  by  the  casein  by  the  percentage  concentration  of  the 
casein  we  obtain  the  number  of  gram  equivalents  of  KOH 
neutralized  by  1  gram  of  casein  at  absolute  neutrality.  The 
following  are  the  results  computed  from  the  data  tabulated 
above: 


Concentration  of  casein,  per  cent 

Gram  equiv.  of  KOH  neutralized 
by  1  gram  of  casein  at  absolute  neu- 
trality at  30  degrees 

0.5 
1.0  (first  series) 
1.0  (second  series) 
1.5 
2.0 
3.0 

52X10-6 
50X10~5 
43X10-5 
53X10-5 
54X10-* 
56X10-5 

Average     51X1Q-5 

The  values  of  the  combining  capacities  of  the  casein  at  neutrality 
to  litmus  computed  in  this  manner  are  seen  to  be  appreciably 
constant.  The  average  value  is  51  X  10~5  equivalents  per  gram 
of  casein,  which  is  in  excellent  agreement  with  that  obtained  by 
fcitration,  namely,  50  X  10~5  equivalent  gram  molecules  per  gram. 
Solutions  of  ovomucoid  require  the  addition  of  a  small  quantity 
of  add  to  obtain  an  absolutely  neutral  solution. f  The  amount 

*  It  was  at  first  thought  necessary  to  pa~s  the  curve  y  =  ax?  +  bxz  +  ex  + 
d,  through  four  of  the  points  of  the  experimental  curve  TT  =  f(bi)  and  to  deter- 
mine algebraically  the  point  of  intersection  of  this  curve  with  the  curve 
y  =  4107  —  0.060  logio  %',  this  was,  however,  found  to  be  an  unnecessary 
refinement,  as  the  results  obtained  did  not  differ  appreciably  from  those 
obtained  by  the  simpler  method  described  above. 

t  In  this  connection  attention  may  be  called  to  the  fact,  which  is  revealed 
in  the  above  tables,  that  when  1  per  cent  ovomucoid  is  dissolved  in  6.95  X 
10"6  N  acid,  although  the  ovomucoid  neutralizes  a  little  of  this  acid,  yet  this 
solution  is  actually  more  acid  than  the  solution  which  is  obtained  by  dissolving 
1  per  cent  ovomucoid  in  0.0005  N  HC1.  A  similar  phenomenon  was  observed 
by  W.  B.  Hardy  in  solutions  of  serum  globulin  (22).  It  is  possible  that  this 
indicates  a  slight  decomposition  of  the  salt  which  was  present  in  these  solu- 
tions by  the  protein,  or,  more  probably  that  it  is  due  to  a  small  proportion  of 
the  free  protein  ionizing  according  to  the  equation  R.COOH  =  RCOO'  +  H+. 


206  ELECTROCHEMISTRY 

of  HC1  bound  by  ovomucoid  at  absolute  neutrality,  estimated 
in  the  above  manner,  is  7  X  10~5  equivalents  per  gram. 

3.  The  Non-Dependence  of  the  Composition  of  the  Compounds 
of  Protein  with  Acids  and  Bases  upon  the  Dilution  of  their 
Solutions.  —  The  view  would  appear  to  be  very  generally  held 
(35)  (9)  (51)  that  the  salts  which  proteins  form  with  acids  and 
bases  are  subject  in  a  very  high  measure  to  hydrolytic  dissoci- 
ation. This  view  is  nevertheless  erroneous.  When  a  base  unites 
with  an  organic  acid  to  form  a  salt  by  the  neutralization  of  a 
— COOH  group  the  reaction  may  be  expressed  as  follows: 

R.COOH  +  KOH  <±  RCOOK  +  H.OH. 

If  the  acid  is  a  very  strong  one  then  the  reaction  will  proceed 
completely  from  left  to  right  and  the  salt  will  not  be  appreciably 
decomposed  by  dilution.  If,  however,  the  acid  is  tolerably  weak 
(e.g.,  acetic  acid)  so  that  the  water  itself,  acting  as  "an  acid,  is 
able,  if  present  in  large  amounts,  to  displace  it  partially  from  its 
combination  with  the  base,  then  the  reaction  will  not  proceed 
completely  from  left  to  right  but  will  pause,  in  accordance  with 
the  mass  law,  in  a  condition  of  balance,  which  is  shifted  towards 
the  right  by  the  addition  of  excess  of  base  and  towards  the  left 
by  dilution.  On  adding  excess  of  base  to  a  dilute  solution  of  an 
acid  of  this  type,  therefore,  more  of  the  base  will  be  observed 
to  be  neutralized,  while  the  addition  of  water  will  result  in  partial 
decomposition  of  the  salt.  Similar  considerations  hold  good,  of 
course,  for  the  salts  of  weak  bases.  When  one  examines  the 
evidence  which  has  been  brought  forward  by  different  observers 
in  support  of  the  thesis  that  protein  salts  are  subject  to  hydrolytic 
dissociation  one  finds  that  it  is  all  of  the  first  kind,  that  is,  con- 
sists in  the  fact  that  upon  the  addition  of  more  acid  or  base  to  the 
protein  solution,  or  of  more  protein,  more  of  the  acid  or  base  is 
bound.  From  this  the  somewhat  illogical  assumption  has  been 
made  that  the  protein  salts  must  exhibit  the  other  characteristic 
property  of  salts  of  weak  acids  and  bases,  namely,  decomposa- 
bility  by  water.  We  have,  however,  seen  that  the  fact  that  pro- 
tein will  bind  more  of  an  acid  or  a  base  in  the  presence  of  an 

This  mode  of  dissociation  would,  of  course,  be  prevented  by  dissociation  of  the 
molecule  through  its  —  N.HOC—  groups,  and,  since  this  is  brought  about 
by  the  addition  of  acid,  the  addition  of  small  quantities  of  acid  might  well 
decrease  the  acidity  (H+  concentration)  of  the  solution. 


NON-DEPENDENCE  OF  COMPOSITION  ON  DILUTION     207 

excess  of  these  reagents  admits  of  a  very  different  explanation, 
and  the  data  cited  above  also  enable  us  to  conclude  that  water 
is  not  able  to  appreciably  decompose  the  potassium  salt  of  casein. 
We  have  seen  that  whether  the  percentage  of  casein  in  solution 
is  0.5  or  3.0  or  intermediate  between  these,  the  quantity  of  potas- 
sium hydrate  which  is  bound  by  a  given  weight  of  casein  at 
absolute  neutrality  is  the  same.  The  dilution  (relative  mass  of 
water  to  salt)  may  vary  600  per  cent  and  yet  the  salt  remains 
unaltered;  it  is  not  decomposed  by  the  water  in  any  preceptible 
degree.  This  result  has  also  been  obtained  for  other  bases, 
including  comparatively  weak  bases  such  as  Ca(OH)2,  by  the 
observers,  cited  in  the  previous  section,  who  have  employed  the 
method  of  direct  titration  and  these  observers  have  also  shown 
that  at  neutrality  to  phenolphthalein  the  composition  of  the 
casein  salt  (=  80  X  10~5  equivalents  of  base  per  gram)  is  always 
the  same  no  matter  what  the  dilution  of  the  system.  Moreover, 
as  the  curves  in  the  figure  on  p.  200  clearly  reveal,  the  compo- 
sition of  the  casein  salt,  when  the  casein  is  exerting  its  maximum 
combining-capacity,  is  the  same  for  very  different  concentrations 
of  this  salt.  That  this  should  be  so  in  alkaline  solution  is  not 
surprising,  since  the  excess  of  alkali  might  be  expected  to  drive 
the  reaction: 

R.COOH  +  KOH  <=>  RCOOK  +  H.OH 

over  towards  the  right;  but  that  it  is  so  in  absolutely  neutral 
solution,  when  the  concentration  of  free  KOH  is  evanescently 
small,  is  an  extremely  striking  fact. 

But,  not  only  is  the  composition  of  the  caseinate  of  potassium 
independent  of  its  dilution  in  neutral  solution,  it  is  also  inde- 
pendent of  its  dilution  in  a  solution  which  is  pronouncedly  acid 
in  reaction.  It  will  be  shown  in  the  succeeding  chapter  that  the 
amount  of  a  base  which  is  bound  by  casein  at  "saturation"  of  the 
base  with  casein,  that  is,  when  there  is  just  sufficient  base  to  hold 
the  casein  in  solution  is  also  independent  of  the  dilution  of  the 
system.  Now  in  this  solution  the  acidity  is  about  10~5  H+ 
(cf.  the  table  of  reactions  to  indicators  on  p.  91).  A  glance  at 
the  formula 

R.COOH  +  KOH<=±R.COOK  +  H.OH 

suffices  to  show  that  in  acid  solutions,  when  the  KOH  concen- 
tration is  excessively  small  and  the  H+  concentration  large  the 


208  ELECTROCHEMISTRY 

tendency  to  hydrolysis  of  a  salt  of  the  type  R.COOK  must  be 
exceptionally  great,  yet  potassium  caseinate  does  not  reveal 
this  tendency,  and  the  conclusion  is  forced  upon  us  that  potas- 
sium caseinate  is  not  formed  in  accordance  with  an  equation  of 
the  above  type. 

Reverting,  now,  to  the  hypothesis  which  I  have  advanced 
regarding  the  mode  of  formation  of  protein  salts,  we  shall  see 
that  in  the  equation  : 

H 

I  +4- 

-N.HOC-  H-  KOH  <=±  N>  -  +  KOC- 

I 
OH 

no  water  is  involved,  and,  consequently,  the  composition  of  the  salt 
must  be  dependent  only  upon  the  relative  concentrations  of  protein 
and  base  and  not  upon  the  total  dilution.  Similarly  in  the  forma- 
tion of  salts  with  acids: 

H 

I  ++ 

-N.HOC-  +  HC1<=±  -N^+  HOC- 


Cl 

no  water  enters  into  the  reaction.  Hence  both  the  dependence 
of  the  composition  of  the  salt  upon  the  excess  of  acid  or  base 
and  its  non-dependence  upon  dilution  receive  a  simple  interpre- 
tation in  the  light  of  this  hypothesis. 

It  will  I  think  be  clear  from  the  foregoing  discussion,  that  the 
non-dependence  of  the  composition  of  protein  salts  upon  their 
dilution  is  one  of  the  most  emphatic  proofs  we  possess  of  the 
fact  that  terminal  —  COOH  and  —  NH2  groups  are  not  respon- 
sible for  their  formation. 

An  experiment  which  demonstrates  in  a  very  striking  manner  the 
fact  that  protein  salts  do  not  undergo  an  appreciable  amount  of 
hydrolytic  dissociation  in  solution  in  water,  nor  split  off  the  inor- 
ganic radical  as  an  ion,  is  the  following.  It  will  be  recollected  that 
casein,  deprived  of  its  combined  base  or  acid,  is  insoluble,  and  that 
if,  to  a  solution  of  a  caseinate  of  a  base,  exactly  enough  free  acid 
be  added  (e.g.,  HC1)  to  completely  neutralize  the  combined  base 
the  free  casein  is  entirely  precipitated.  Now  one  gram  of  ovo- 
mucoid  (60)  combines  with  45  X  10~5  equivalents  of  HC1  to  form 


"  ISOELECTRIC  "   CONDITION  209 

a  compound  such  that  less  than  1  per  cent  of  the  acid  remains 
uncombined  (as  estimated  by  gas-chain  measurements).  One 
gram  of  casein  combines  with  90  X  10~5  equivalents  of  KOH  to 
form  a  compound  such  that  less  than  ^  per  cent  of  the  KOH 
remains  uncombined  (59).  If,  now,  these  salts  were  appreciably 
subject  to  hydrolytic  dissociation,  or  even  if  they  yielded  Cl'  and 
H+  ions  respectively,  then  on  mixing  two  volumes  of  a  solution 
of  the  ovomucoid  salt  with  one  volume  of  a  solution  of  the  casein 
salt  (each  of  the  same  percentage  concentration)  the  K+  pro- 
vided by  the  caseinate  would  be  exactly  neutralized  by  the  Cl' 
provided  by  the  ovomucoid  salt  and  it  might  be  anticipated 
that  free  uncombined  casein  would  be  precipitated.  Nothing 
of  the  sort  occurs,  however.  If  to  25  cc.  of  a  2  per  cent  solution 
of  the  casein  salt  are  added  50  cc.  of  a  2  per  cent  solution  of  the 
ovomucoid  salt  the  mixture  is  no  more  opalescent  than  its  con- 
stituent parts  and  the  conductivity  of  the  mixture  is  the  sum  of  the 
separate  conductivities  of  the  two  protein  salts*  If  the  mixture 
be  allowed  to  stand  in  the  presence  of  toluol  for  a  considerable 
period  at  36  degrees,  however,  after  24  to  45  hours  a  marked 
increase  in  its  opalescence  is  observed;  after  two  or  three  days 
traces  of  casein  begin  to  be  deposited,  and  after  three  to  four 
days  all  of  the  casein  is  found  to  have  been  precipitated.  The 
precipitation  of  the  casein  is  accompanied  by  a  marked  increase 
in  the  conductivity  of  the  mixture,  attributable  to  the  setting 
free  of  KC1.  It  is  therefore  evident  that  at  the  beginning  the 
mixture  must  contain  only  minute  traces  of  K+  and  Cl'  ions  and 
that  the  protein  salts  only  yield  up  these  ions  with  extreme 
slowness. 

4.  The  "Isoelectric"  Condition  of  Proteins  at  Certain  H+  and 
OH'  Concentrations.  —  If  we  assume  that  the  proteins  dissociate 
H+  and  OH'  ions  according  to  the  formula: 

XNH3OH^     /NH3OH 

NCOOH  '        NCOO' 
and 

/NH3OH 


R  ^R  +  OH' 

NCOOH  NCOOH 

*  Conductivity  of  a  0.5  per  cent  solution  of  potassium  casemate  containing 
90  X  10~5  equivalents  of  KOH  per  gram  at  30  degrees  equals  38.0  X  10~6 
reciprocal  ohms.  Conductivity  of  a  1  per  cent  solution  of  ovomucoid  chloride 


210  ELECTROCHEMISTRY 

then  the  dissociation  of  H+  ions  must  be  inhibited  and  that  of 
OH'  ions  encouraged  by  the  presence  of  an  excess  of  H+  ions, 
while  that  of  OH'  must  be  inhibited  and  of  H+  ions  encouraged 
by  an  excess  of  OH'  ions,  and  a  reaction  must  exist  at  which 
H+  and  OH'  are  split  off  in  exactly  equal  quantities  so  that  the 
salt  is  "isoelectric,"  i.e.,  wanders  equally  in  both  directions  in  an 
electric  field.  A  simple  calculation  suffices  (53)  to  show  that  if 
the  mode  of  dissociation  of  the  protein  is  actually  that  repre- 
sented by  the  above  formulae,  then  the  number  of  protein  ions 
in  a  solution  of  proteins  must  attain  a  minimum  in  solutions  in 
which  it  is  "isoelectric." 

A  number  of  observations,  to  which  more  detailed  reference 
will  be  made  in  a  later  chapter,  tend  to  establish  the  fact  that  the 
viscosity  of  protein  solutions  is  in  the  greater  proportion  attributa- 
ble to  its  ions,  and  that,  consequently,  when  the  ionization  of  a 
protein  is  at  a  minimum  the  viscosity  of  its  solution  will  tend 
to  a  minimum  also. 

Applying  these  considerations,  and  also  employing  direct  obser- 
vation of  the  movement  of  the  protein  in  an  electric  field,  Michaelis 
and  co-workers  (40)  (41)  (42)  (43)  (44)  (45)  have  endeavored 
to  ascertain  the  hydrogen  ion  concentration  of  the  solution  in 
which  egg-albumin  is  isoelectric. 

In  the  light  of  the  view  which  I  have  developed  concerning 
the  mode  of  formation  and  dissociation  of  the  protein  salts  a 
somewhat  different  interpretation  must  be  placed  upon  these 
results.  A  glance  at  the  figure  on  p.  185  will  show  that  if  the 
protein  is  soluble  in  the  free  condition  and  uncombined  with 
bases  or  acids  it  must,  if  it  is  ionized  at  all,  migrate  equally  in 
both  directions  through  the  solution  under  the  influence  of  an 
electric  current,  reacting  at  the  electrodes  as  follows : 


(Anode)  2  HOOC.R.N^  =  2  HOOC.R.NH2  +  02 
I 
OH 

(Cathode)  H2N.R.COH  +  H2O  =  H2N.R.COOH  +  H2 

containing  45  X  10~5  equivalents  of  HC1  per  gram  at  30  degrees  equals  80.1  X 
10~5  reciprocal  ohms.  Sum  of  these  conductivities  equals  118.1  X  10~6  recip- 
rocal ohms.  Conductivity  of  a  mixture  containing  the  two  salts  in  the  above 
concentrations  at  30  degrees  equals  108.5  X  10~5  reciprocal  ohms. 


BIOLOGICAL  APPLICATIONS  211 

and  regenerating  the  free  protein  to  again  participate  in  carrying 
the  current  both  ways.  Provided,  therefore,  the  two  ions  are 
of  nearly  equal  weight  (and  we  have  seen  that  in  the  case  of  the 
ions  of  potassium  caseinate  the  ions  are  nearly  equal  in  weight, 
cf.  previous  chapter)  no  noticeable  change  in  the  distribution 
of  the  protein  in  the  electric  field  will  be  observed.  If  the  ions 
should  be  of  unequal  weight,  then  a  trace  of  acid  or  alkali  com- 
bined with  the  protein  might  suffice  to  neutralize  this  inequality 
and  render  the  protein  apparently  "isoelectric."  The  "iso- 
electric" protein  of  Michaelis  is,  therefore,  in  all  probability, 
merely  free  protein  uncombined  with  acids  or  bases.  This  view 
of  Michaelis'  result  is  supported  by  Pauli  and  Wagner  (50)  who 
point  out  that  the  egg-albumin  employed  by  Michaelis  is,  in 
reality,  an  albuminate  of  a  base,  which  therefore  requires  the 
addition  of  some  acid  to  set  the  protein  free.  The  free  protein 
is  sparingly  ionized  and  hence  the  viscosity  of  its  solution  is  low. 

5.  Biological  Applications;  the  Neutrality  of  the  Tissues  and 
of  the  Tissue  Fluids.  —  The  power  which  the  protein  salts 
possess  of  neutralizing  additional  equivalents  of  acid  or  base  by 
the  opening  up  of  fresh  —  N.HOC—  bonds  confers  upon  them, 
since  the  reaction  of  free  protein  is  usually  nearly  neutral,  a 
remarkable  power  of  maintaining  the  neutrality  of  their  solu- 
tions, and  it  appears  probable  that  this  characteristic  of  the 
protein  salts  plays  an  important  part  in  maintaining  the  neu- 
trality of  the  tissues  and  a  not  insignificant  part  in  maintaining 
the  neutrality  of  the  tissue  fluids. 

The  older  statements  which  are  found  in  physiological  and 
medical  literature  concerning  the  reactions  of  the  blood  are  totally 
unreliable,  since  they  were  based  upon  the  erroneous  belief  that 
it  is  possible  to  ascertain  the  reaction  of  a  fluid  such  as  the  blood 
by  titration.  The  determinations  of  Hoeber  (31),  Farkas  (10) 
(11)  (12)  (13),  Fraenkel  (18),  Szily  (69)  (70),  Hasselbalch  (23) 
(24)  and  de  Corral  (8)  carried  out  by  the  potentiometric  method, 
have  shown  that  the  H+  and  OH'  ion  concentrations  of  normal 
blood  are  very  close  to  those  at  neutrality  (neutral  point  = 
8  X  10~8  H+  and  OH'),  the  alkalinity  first  found  by  Hoeber  having 
been  traced  to  the  fact  that  the  stream  of  hydrogen,  used  to 
impregnate  the  platinum  electrode  of  the  gas-chain,  washed  the 
CO2  out  of  the  blood.  On  eliminating  this  source  of  error  the 
blood  is  found  to  be  almost  absolutely  neutral,  the  exact  OH' 


212  ELECTROCHEMISTRY 

concentration  in  normal  blood  at  physiological  C02  pressures 
(0.028  to  0.054  atmosphere)  being,  according  to  recent  deter- 
minations, about  0.37  X  10~7. 

Friedenthal  (19)  and  von  Szily  (69),  employing  a  somewhat 
different  method  of  investigation,  have  reached  the  same  con- 
clusion as  the  above  quoted  observers.  They  utilized  the  indi- 
cator-method of  Friedenthal,  Fels  and  Salm.  Mixtures  of  acid 
and  basic  phosphates  of  known  H+  and  OH'  content  (determined 
by  Salm,  using  the  potentiometric  method)  were  tinged  with  a 
number  of  different  indicators  and  the  samples  of  the  body- 
fluid  under  examination  were  tinged  with  equal  quantities  of 
the  same  indicators  —  the  colors  of  the  two  series  of  fluids  were 
then  compared.  The  phosphate-mixture  which  approximated 
most  closely  in  its  indicator  reactions  to  the  normal  blood-serum 
had  an  acidity  corresponding  to  6.5  X  10~8  H+,  absolute  neutrality 
being  8  X  10~8  H+. 

Moreover  the  investigations  of  Friedenthal  (19)  (20)  and  of 
Foa  (15)  (16)  (17)  have  shown  that  not  only  is  the  blood  almost 
exactly  neutral  but  that  almost  all  of  the  tissue-fluids  are  also 
approximately  neutral.  Thus,  the  pancreatic  juice  of  a  dog,  one 
of  the  most  alkaline  of  the  body-fluids,  contained  5  X  10~9  H+, 
corresponding  to  an  alkalinity  of  13  X  10~7  OH'.  Hitherto,  ac- 
cording to  Friedenthal  (20),  no  animal  fluid  has  been  found  con- 
taining less  than  10~10  N  H+,  that  is,  more  than  about  6  X  10~5 
N  OH'.  The  gastric  juice  of  a  dog  was,  however,  found  to  be 
1000  times  more  acid  than  the  pancreatic  juice  is  alkaline,  that 
is,  it  possessed  the  acidity  of,  approximately,  a  hundredth  normal 
solution  of  HC1. 

With  few  exceptions,  therefore,  the  tissue  fluids  are  practically 
neutral  in  reaction.  It  is  a  significant  fact  also  (Friedenthal 
(20),  Loeb  (37))  that  the  naturally  occurring  waters,  in  which 
the  lives  of  aquatic  animals  are  spent,  are  also  very  nearly  neutral 
in  reaction.  "We  may  therefore  draw  the  conclusion  that  life- 
phenomena  occur  in  a  neutral  liquid"  (Loeb,  loc.  cit.),  and,  more- 
over, save  in  certain  special  localities,  such  as  the  internal  wall 
of  the  stomach,  the  life-process  occurs  exclusively  in  a  neutral 
milieu;  thus  even  an  increase  of  50  per  cent  in  the  minute  H+ 
concentration  of  normal  blood  induces  profound  toxic  symptoms, 
and  an  increase  to  1  X  10~7  N  H+  induces  fatal  acidosis. 

In  view  of  the  fact  that  the  products  of  metabolism  include  a 


BIOLOGICAL  APPLICATIONS  213 

large  proportion  of  acid  substances,  the  mechanism  which  pre- 
serves the  neutrality  of  the  tissues  and  tissue-fluids  despite  the 
relatively  enormous  daily  and  hourly  fluctuations  in  acid-pro- 
duction or  consumption  which  necessarily  occur  within  the  body, 
is,  in  all  probability,  one  of  prime  physiological  importance. 

The  acid  which  is  produced  most  copiously  in  the  body  is 
carbonic  acid.  The  classic  theory  regarding  the  neutralization 
of  this  acid  is  that  of  Fernet  (14),  Heidenhain  and  Mayer  (25) 
(26)  and  von  Bunge  (6)  (7)  who  supposed  that  it  is  bound  by  the 
disodium  phosphate  in  the  blood,  according  to  the  balanced 
reaction  : 

C02  +  H20  +  Na2HP04  ?±  NaHC03  +  NaH2P04, 


the  reaction  proceeding  towards  the  right  in  the  tissues,  where 
the  C02  tension  is  high,  towards  the  left  in  the  lungs,  where  the 
C02  tension  (active  mass  of  CO2)  is  low.  Sertoli  (65),  however, 
and  Mroczkovsky  (46)  pointed  out  that  the  quantity  of  phos- 
phoric acid  in  the  blood  of  many  animals,  e.g.,  the  ox  and  the 
pig,*  is  too  small  to  play  the  important  r61e  assigned  to  it  by 
these  investigators. 

We  have  seen  that  the  alkali-  and  acid-equivalent  of  the  pro- 
teins varies  very  markedly  with  the  hydroxyl-  or  hydrogen- 
contents  of  their  solutions.  Thus  one  gram  of  serum-globulin 
(Cf.  Chap.  V)  in  solutions  neutral,  or  approximately  neutral,  to 
litmus  (absolute  neutrality:  H+  =  OH'  =  8  X  10~8)  neutralizes 
10  X  10~5  equivalent  gram  molecules  of  a  base,  while  in  solutions 
neutral  to  phenolphthalein  (OH7  =  20  X  10~7)  1  gram  neutralizes 
20  X  10~5  equivalent  gram  molecules  of  a  base.  The  significance 
of  these  figures  may,  perhaps,  be  more  fully  realized  when  stated 
thus:  in  one  litre  of  NaOH  solution  containing  1  per  cent  of 
globulin  and  neutral  to  litmus,  addition  of  100  X  10~5  gram- 
equivalents  of  NaOH  only  raises  the  alkalinity  (hydroxyl-content) 
of  the  solution  by  about  0.2  X  10~5,  that  is,  by  two-tenths  of  a 
per  cent  of  the  change  in  the  sodium  content  of  the  solution. 
Casein,  at  neutrality  to  litmus,  neutralizes  50  X  10~5  gram- 
equivalents  of  base  per  gram,  and  at  "saturation,"  i.e.,  at  an 
acidity  of  about  50  X  10~7  H+,  it  neutralizes  only  11.4  X  10~5 
gram-equivalents  of  base  per  gram.  Hence  to  a  litre  of  a  1  per 
cent  solution  of  sodium  caseinate,  neutral  to  litmus,  we  should 
*  Cf  .  von  Bunge  (7)  . 


214 


ELECTROCHEMISTRY 


have  to  add  400  X  10~5  equivalents  of  HC1  to  reach  an  acidity 
of  0.5  X  10~5  equivalents  —  just  over  one-tenth  of  a  per  cent  of 
the  acid  added.  Were  the  casein  present  in  a  concentration  of 
8  per  cent  (the  percentage  concentration  of  the  proteins  in  the 
blood),  3200  X  10~5  gram  equivalents  of  HC1,  or  320  cc.  of  a 
tenth  normal  solution,  would  have  to  be  added  to  a  litre  of  the 
solution  to  attain  an  acidity  of  0.5  X  10~5  equivalents.  One 
twentieth  of  a  cubic  centimeter  of  tenth  normal  HC1,  added  to  a 
litre  of  pure  distilled  water,  would  produce  the  same  acidity.  The 
power  of  the  proteins  to  maintain  the  neutrality  of  the  fluids  in 
which  they  occur  is  therefore  very  remarkable,  and  Loeb  (36), 
myself  (55)  (56)  (57)  (58)  and  others  have  suggested  that  they 
play  an  important  part  in  regulating  the  neutrality  of  the  tissues 
and  tissue-fluids,  while  Hoppe-Seyler  (32),  Sertoli  (65),  Zuntz  (72) 
(73)  and  myself  (55)  (57)  have  advanced  the  view  that  the  liber- 
ation of  C02  from  the  blood  in  the  lungs  is  accompanied  by  a 
transport  of  sodium  from  the  carbonic  acid  to  the  proteins  of  the 
plasma. 

Friedenthal  (19)  (20),  Spiro  (68),  Loeb  (36)  and  Henderson 
(27)  (28)  (29)  (30)  have  pointed  out  that  the  bicarbonates  of  the 
blood  and  other  media  in  which  organisms  live  must  also  be  of 
importance  in  preserving  their  neutrality.  Henderson  points  out 
that  the  rate  of  change  in  the  alkalinity  or  acidity  of  a  solution 
of  an  acid  is  a  minimum  when  the  dissociation  constant  of  the 
acid  is  equal  to  the  hydrogen  ion  concentration  (8  X  10~8  N)  at 
neutrality.  He  illustrates  this  by  the  following  table,  showing 
the  amount  of  tenth  normal  alkali  required  to  secure  a  definite 
but  arbitrarily  chosen  change  in  alkalinity  when  added  to  equal 
amounts  of  the  undermentioned  acids. 


Acid 

Dissociation-con- 
stant X  10~7 

Cubic  centimeters 
of  alkali  required 

Phenol     

0.0013 

0.01 

Boric  acid. 

0  017 

0  08 

Hydrogen  sulphide 

0  57 

1  10 

Mono-sodium  phosphate 

2  0 

1.00 

Carbonic  acid 

3  0 

0.72 

Picolinic  acid                            

18.0 

0.10 

Acetic  acid   .                        

180.0 

0.03 

Henderson  believes  that  the  neutrality  of  the  tissue  fluids  is 
chiefly   maintained    by    the    bicarbonates    which    they    contain 


BIOLOGICAL  APPLICATIONS  215 

and  in  a  minor  degree  by  the  phosphates  and  by  the  proteins. 
He  illustrates  his  hypothesis  by  reference  to  a  system  tenth- 
molecular  in  total  carbonic  acid  and  equally  concentrated  in 
total  phosphoric  acid,  combined  or  uncombined  with  sodium, 
and  he  shows  that  in  order  to  appreciably  change  the  acidity  of 
this  system  a  quantity  of  acid  comparable  with  the  total  amount 
of  sodium  which  is  present  at  neutrality  must  be  added.  He 
estimates,  from  known  quantitative  data,  the  quantity  of  acid 
which  would  be  neutralized  by  the  bicarbonates  of  the  blood  in 
passing  from  the  reaction  of  normal  blood  (=  0.37  X  10~7  H+  at 
38  degrees)  to  that  of  blood  in  advanced  acid  intoxication  (about 
1.00  X  10~7  H+  at  38  degrees).  At  the  same  time,  from  data 
derived  from  experiments  in  which  he  employed  indicators  to 
determine  the  change  in  the  reaction  of  solutions  of  the  serum- 
proteins  to  which  varying  amounts  of  acid  and  alkali  had  been 
added,  he  estimated  the  amount  of  acid  which  is  neutralized  by 
the  proteins  of  the  blood,  and  their  salts,  as  the  reaction  of  their 
solutions  changes  by  the  same  amount,  and  he  concludes  that 
it  cannot  be  more  than  one-fifth  of  the  amount  of  acid  which 
would  be  neutralized  by  the  bicarbonates. 

Experimental  results  which  involve  the  use  of  indicators  to 
determine  such  slight  changes  in  reaction  as  these  afford  an  un- 
satisfactory basis  for  so  important  a  theoretical  conclusion.  Ac- 
cordingly, employing  the  potentiometric  method,  I  carried  out  a 
series  of  determinations  of  the  reactions  of  solutions  of  the  pro- 
teins of  serum,  of  the  concentration  in  which  they  occur  in  serum, 
to  which  varying  amounts  of  acid  or  alkali  had  been  added  (58). 
The  results  of  these  experiments  entirely  confirm  Henderson's 
estimate  of  the  neutralizing  power  of  the  proteins  of  blood-serum 
between  the  reactions  mentioned  above. 

The  proteins  of  ox-blood  serum  were  coagulated  by  alcohol, 
carefully  washed  with  alcohol  and  ether  and  dried.  They  were 
then  dissolved  in  0.01  N  KC1  containing  varying  concentrations 
of  KOH  or  HC1,  each  solution  containing  8  per  cent  of  the  pro- 
teins (=  percentage  concentration  in  blood).  The  following  were 
the  results  obtained.* 

*  The  value  of  K,  the  constant  ionic  product  for  water  at  34  degrees,  was 
taken  as  (1.47  X  lO"7)2  =  2.16  X  10~14  (Cf.  Kohlrausch  and  Heydweiller 
(33) ) .  The  experimental  error  in  the  determination  of  CH+  in  the  "  unknown  " 
is  not  more  than  6  per  cent  of  CH+. 


216 


ELECTROCHEMISTRY 


Known  element  of 
chain 

"Unknown"  element  of 
chain 

Potential  of 
chain,  volt 

CH+  in  "un- 
known" ele- 
ment 

[Log10  CH+  in 
"unknown" 
element 

0.01  ATKOH 

0.01ATKOH+8per 
cent  serum  pro- 
teins. 

0.0920 

7.35X10-11 

-10.13 

0.01JVKOH 

0.005  ATKOH+8  per 
cent  serum  pro- 
teins. 

0.1186 

2.03X10-10 

-  9.79 

0.01  N  KOH 

8  per  cent  serum 
proteins. 

0.1710 

1.  52X10-9 

-  8.82 

0.01ATKOH 

0.005  N  HC1+8  per 
cent  serum  pro- 
teins. 

0.2277 

1.  33X10-8 

-  7.88 

0.01  N  HC1 

0.01  AT  HC1+8  per 
cent  serum  pro- 
teins. 

0.2968 

1.15X10-7 

-  6.94 

0.01  ATHC1 

0.02  N  HC1+8  per 
cent  serum  pro- 
teins. 

0.2198 

2.20X10-* 

-  5.66 

If,  taking  the  solution  of  8  per  cent  serum-proteins  in  neutral 
0.01  N  KC1  as  the  zero-point  on  the  axis  of  the  abscissae,  we 
measure  off  to  the  right  of  this  point  the  concentrations  of  acid 
in  which  the  proteins  were  dissolved,  and  to  the  left  of  this  point 
the  concentrations  of  alkali  in  which  they  were  dissolved  and 
plot  the  values  of  log  CH+  given  in  the  above  table,  we  obtain 
a  curve,*  by  interpolation  from  which  it  is  readily  found  that  in 
passing  from  the  reaction  0.37  X  10~7  H+  to  the  reaction  1.00  X 
10~7  H+,  100  cc.  of  an  8  per  cent  solution  of  the  serum  protein 
neutralizes  2.25  cc.  of  JV/10  HC1,  that  is  22.5  cc.,  or  about  one- 
fifth  of  its  own  volume  of  N/WO  HC1.  According  to  Henderson, 
in  passing  through  the  same  range  of  H+  concentrations  the 
bicarbonates  in  100  cc.  of  blood  will  neutralize  the  equivalent 
of  100  cc.  of  N/WQ  HC1.  Hence  we  must  conclude  that,  between 
the  reactions  mentioned,  the  proteins  of  the  serum  are  only  one- 
fifth  as  efficient  in  maintaining  its  neutrality  as  the  bicarbonates. 
To  the  neutralizing  power  of  the  serum  proteins  must  be  added, 
in  circulating  plasma,  that  of  the  fibrinogen.  This  is,  however, 
probably  very  slight,  since  only  a  very  small  percentage  of  fibrin- 
ogen is  contained  in  the  blood. 

*  For  the  form  of  the  curve  see  my  original  communication  (58). 


BIOLOGICAL  APPLICATIONS  217 

It  has  been  shown  by  Marshall  (39)  that  the  major  part  of  the 
neutralizing  power  of  saliva  is  likewise  attributable  to  its  inor- 
ganic constituents. 

It  would  not  be  safe,  however,  to  conclude  from  these  results 
that  the  part  played  by  the  proteins  in  maintaining  the  neutrality 
of  the  tissues  is  not,  possibly,  equal  in  magnitude  to,  or  even 
greater  than,  that  played  by  the  bicarbonates  and  acid  phosphates. 
Thus,  referring  to  the  tables  in  section  2  it  will  be  seen  that 
100  cc.  of  a  1.5  per  cent  solution  of  potassium  caseinate,  in  pass- 
ing from  the  reaction  0.34  X  10~7  H+  to  the  reaction  1.14  X  10~7 
H+  neutralizes  the  equivalent  of  25  cc.  of  N/ 10  HC1;  a  3  per 
cent  solution  (the  concentration  of  casein  in  cow's  milk)  would 
therefore  neutralize,  in  passing  from  the  former  to  the  latter  of 
the  above  reactions,  the  equivalent  of  one-half  its  volume  of 
N / 100  HC1.  The  part  played  by  the  casein  of  milk  in  main- 
taining the  neutrality  of  this  tissue-fluid  must  therefore  be  of 
very  considerable  importance.  In  the  tissues,  not  only  are  the 
proteins  more  concentrated  than  they  are  in  the  tissue-fluids, 
but,  owing  to  the  predominance  of  nucleo-proteins  which  have 
a  high  combining-capacity  for  bases,  their  power  of  maintaining 
the  neutrality  of  the  solutions  is  not  improbably  even  higher 
than  that  of  the  majority  of  the  proteins  which  occur  in  the 
tissue-fluids.  We  are  not,  at  present,  in  the  possession  of  any 
data  which  would  render  an  estimate  of  the  relative  importance 
of  these  factors,  for  the  maintenance  of  the  neutrality  of  the 
tissues  themselves,  in  the  slightest  degree  reliable. 

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(56)  Robertson,  T.  Brailsford,  "The  Proteins,"  Univ.  of  California,  Publ. 

Physiol.  (1909). 

(57)  Robertson,  T.  Brailsford,  Ergeb.  d.  Physiol.  10  (1910),  p.  293. 

(58)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  7  (1910),  p.  351. 

(59)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  14  (1910),  p.  528. 

(60)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  14  (1910),  p.  709. 

(61)  Rohonyi,  H.,  Biochem.  Zeit.  44  (1912),  p.  165. 

(62)  Salm,  E.,  Zeit.  f.  physikal.  Chem.  57  (1906),  p.  471. 

(63)  Schmidt,  C.  L.  A.,  Journ.  Biol.  Chem.  25  (1916),  p.  63. 

(64)  Schmidt,  C.  L.  A.,  Univ.  of  California  Publ.  Pathol.  2  (1916),  p.  157. 

(65)  Sertoli,  E.,  Hoppe-Seyler's  Med.  Chem.  Untersuch.  Berlin  (1868),. 

Heft  3,  p.  350. 

(66)  Soldner,  F.,  Landw.  Versuchsstat.  35  (1888),  p.  351. 

(67)  Sorensen,  S.  P.  L.,  Ergeb.  d.  physiol.  12  (1912),  p.  393. 

(68)  Spiro,  K.,'and  Pemsel,  W.,  Zeit.  f.  physiol.  Chem.  26  (1898),  p.  233. 

(69)  von  Szily,  A.,  Orvosihetilap  (1903),  Nr.  32;   cited  after  Maly's  Jah- 

resber.  f.  Tierchem.  33  (1903),  p.  179. 

(70)  von  Szily,  A.,  Arch.  f.  d.  ges.  Physiol.  115  (1906),  pp.  72  and  82. 

(71)  Van  Slyke,  L.  L.,  and  Hart,  E.  B.,  Amer.  Chem.  Journ.  33  (1905),  p. 

461. 

(72)  Zuntz,  N.,  Hermann's  "Handbuch  der  Physiologic,"  Leipzig  (1880), 

Bd.  4  T.  2. 

(73)  Zuntz,  N.,  Arch.  f.  (Anat.  und)  Physiol.  (1893),  p.  556. 


CHAPTER  X 

THE  ELECTRICAL  CONDUCTIVITY  OF  SOLUTIONS  OF  PROTEIN 

SALTS 

1.  The  Influence  of  Dilution  upon  the  Conductivity  of  Solu- 
tions of  Protein  Salts.  —  The  Ostwald  dilution-law  for  a  binary 
electrolyte  is  usually  formulated  as  follows : 


(-£) 


where  K  is  the  dissociation-constant,  fiv  is  the  molecular  con- 
ductivity at  dilution  V  and  /^  is  the  molecular  conductivity 
at  infinite  dilution,  that  is:  96.44  (u  +  v)  where  u  and  v  are 
average  equivalent  migration  velocities  in  centimetres  per 
second  under  unit  potential  gradient,  of  the  cations  and  anions 
respectively. 

Now  fjLv  =  — ,  where  x  is  the  specific  conductivity  in  reciprocal 

ohms,  and  m  is  the  equivalent  concentration,  i.e.,  y  ;  hence  from 
the  above  equation  we  have 

_/-       1.037  X  10-2   \          /1.037  X  10-2   V 

K[l 7 ; r-X]  =  mi 7 ; r—  X] 

\          m  (u  +  v)      I         \  m  (u  +  v)      / 

which  reduces  to 

1.037  X  10-2      .    1.075  X  10-4    2  ,.. 

m  =  ; X  H ^r-f : rr~   X*.  (l) 

U  +  V  K(U  +  V)Z 

The  same  formula  can  be  derived  as  follows  (8);  let  c  be  the 
equivalent  concentration  of  the  ions,  m  and  K  having  the  same 
significance  as  before;  then,  applying  the  mass  law,  we  have 

C2  =  k  (m  -  c). 

Now  1.037  X  lO-2  x  =  (u  +  v)  c;  hence 
_  1.037  X  10-2 

u  +  v 

substituting  in  the  above  equation  we  regain  equation  (i). 

220 


DILUTION  221 

From  this  mode  of  deriving  the  formula  it  is  clear  that  the 
total  equivalent  concentration  of  the  electrolyte  may  be  expressed 
in  terms  of  the  electric  conductivity  of  its  solution  as  the  sum 
of  two  factors,  the  first,  directly  proportional  to  the  conductiv- 
ity, being  the  equivalent  concentration  of  the  dissociated  part 
of  the  electrolyte  and  the  second,  directly  proportional  to  an 
exponent  of  the  electrical  conductivity  (in  this  instance  2),  be- 
ing the  undissociated  part  of  the  electrolyte. 

The  salts  which  the  proteins  form  with  inorganic  bases  and 
acids  obey  the  Ostwald  dilution-law  for  a  binary  electrolyte  (7) 
(8)  (10)  (12)  (15)  (16)  (17)  (19),  that  is,  the  relationship  between 
the  equivalent  concentration  of  the  protein  salt  and  the  con- 
ductivity (in  reciprocal  ohms  per  cubic  centimetre)  is  that  indi- 
cated by  formula  (i).  It  is  to  be  observed,  however,  that  without 
additional  assumptions  we  do  not  know  what  is  the  equivalent 
concentration  of  a  protein  salt.  If  we  assume,  however,  that  m, 
the  equivalent  concentration  of  the  base  or  acid  which  is  neu- 
tralized by  the  protein,  bears  a  constant  proportion  to  the  true 
equivalent  concentration  of  the  protein  salt,  then  (i)  becomes 

1.037  X  lO-2         1.075  X  10~4  , 
m  = — —      x  _|_  _^_^ — —     X2  (u) 

p  (u  +  v)  Kp  (u  +  v)2 

in  which  m  is  now  the  known  equivalent  concentration  of  the 
base  or  acid  which  is  bound  by  the  protein  and  p  is  the  number 
of  equivalents  of  protein  salt  to  which  each  equivalent  of  neutralized 
add  or  base  gives  rise.  This  equation  may  be  written: 

m  =  Ax  +  Bx2,  (iii) 

in  which  A  and  B  are  constants,  respectively  equal  to 
1.037  X  10-2  1.075  X  1Q-4 

P  (u  +  v)  Kp  (u  +  *)2  ' 

The  fact  that  the  dependence  of  the  conductivity  of  solutions 
of  protein  salts  upon  their  dilution  obeys  the  formula  (iii),  there- 
fore, shows  that  for  a  given  salt  p  is  constant;  in  other  words, 
for  a  given  combination  of  acid  or  base  with  protein,  containing 
a  given  proportion  of  the  acid  or  base,  the  number  of  equivalents 
of  protein  salt  to  which  one  equivalent  of  neutralized  acid  or 
base  gives  rise  is  independent  of  the  dilution. 

The  validity  of  equation  (iii)  as  applied  to  solutions  of  protein 
salts  may  be  gathered  from  the  accompanying  tables.  In  each 
case  the  most  probable  values  of  the  constants  A  and  B  are  com- 


222 


ELECTROCHEMISTRY 


puted  from  all  of  the  experimental  data  by  Gauss'  method  of 
least  squares.  Inserting  these  values  of  the  constants  and  the 
experimental  values  of  x  in  equation  (iii),  the  " calculated" 
values  of  ra  are  computed.  In  the  third  column  of  the  tables 
is  given  the  ''Degree  of  Dissociation"  of  the  salt  calculated  from 
the  ratio  of  Ax  in  equation  3  to  the  " calculated"  value  of  ra. 

The  conductivity  of  the  distilled  water  (usually  not  in  excess 
of  4  X  10~6)  is  subtracted  from  each  of  the  observed  conductivities. 


TABLE  I 

Sodium  Caseinate.     Approx.   neutral   to   Litmus.     Approx.    50  X 
Equivalents  NaOH  per  gram.     Temperature  25  degrees 
A  =  19.51        B  =  9611 


mX10« 
(experimental) 

mX10< 
(calculated) 

Degree  of  dissocia- 
tion (per  cent) 

200 

201 

73 

100 

98 

83 

68 

66 

88 

50 

51 

89 

40 

42 

90 

28 

29 

94 

20 

22 

93 

10 

17 

98 

TABLE  II* 

Ammonium  Caseinate.    Approx.  neutral  to  Litmus.    Approx.  50  X  10~5 
Equivalents  NH4OH  per  gram.    Temperature  25  degrees 
A  =  16.27        B  =  6188 


mX  10* 
(experimental) 

m  X  10* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

200 

201 

74 

100 

99 

84 

68 

67 

88 

50 

50 

90 

40 

40 

93 

28 

30 

93 

20 

24 

96 

10 

11 

99 

*  T.  Brailsford  Robertson  (8).  The  exact  proportion  of  base  to  casein  in  these  experiments  is 
not  certain,  since  the  original  solution  which  was  diluted  to  yield  the  remainder  was  made  up  by 
shaking  up  an  0 . 02  N  solution  of  the  base  with  excess  of  casein  under  the  erroneous  belief  (Cf .  T. 
Brailsford  Robertson  (6)  (8)  (11))  that  the  maximum  amount  of  casein  which  a  given  concentra- 
tion of  a  base  would  dissolve,  when  shaken  up  with  excess  of  casein,  was  that  with  which  it  will 
unite  to  form  the  "neutral"  caseinates;  i.e.,  containing  50  X  1Q-5  equivalents  of  base  per  gram. 
Since,  however,  casein  dissolves  rapidly  up  to  this  point  and  very  slowly  afterwards  the  composi- 
tion of  the  salt  formed  was  probably  very  nearly  that  indicated  at  the  head  of  the  tables.  More- 
over the  solutions  were  tested  and  found  to  be  neutral  to  litmus.  The  last  observation  in  Table  I 
and  the  last  but  one  in  Table  II  are  omitted  in  computing  the  values  of  the  constants. 


DILUTION 


223 


TABLE  III* 

Sodium  Caseinate.     Neutral  to  Phenolphthalein.     80  X  10~5  Equiva- 
lents NaOH  per  gram.    Temperature  25  degrees 
A  =  15.76       B  =  4979 


mX  10* 
(experimental) 

m  X  10* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

250 

250 

73 

125 

132 

77 

63 

62 

89 

31 

33 

94 

16 

18 

96 

TABLE  IV* 

Ammonium  Caseinate.    Neutral  to  Phenolphthalein.    80  X  10~5  Equiva- 
lents NH4OH  per  gram.     Temperature  25  degrees 

A  =  12.60        5  =  3978 


OT  X  10* 

(experimental) 

m  X  10* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

180 

181 

75 

90 

88 

84 

45 

44 

91 

23 

23 

96 

11 

11 

100 

*  The  calculations  in  Tables  III  and  IV  are  based  upon  the  experimental  data  of  Sackur  (21). 
Cf.  T.  Brailsford  Robertson  (10). 


TABLE  V 

Potassium  Caseinate.    Neutral  to  Phenolphthalein.    80  X  10~5  Equiva- 
lents KOH  per  gram.    Temperature  30  degrees 

A  =  12 .41        B  =  1666 


m  X  10* 
(experimental) 

m  X  10« 

(calculated) 

Degree  of  dissocia- 
tion, per  cent 

250 

250 

85 

125 

124 

89 

63 

64 

94 

31 

33 

97 

16 

17 

100 

224 


ELECTROCHEMISTRY 


TABLE  VI 

Calcium  Casemate.     Neutral  to  Phenolphthalein.     80X10"5  Equiva- 
lents Ca(OH)2  per  gram.    Temperature  30  degrees 

A  =  27.89        B  =  26,700 


mX  10* 
(experimental) 

m  X  10* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

320 

320 

26 

240 

243 

29 

160 

151 

35 

120 

115 

39 

80 

81 

44 

60 

66 

48 

40 

47 

53 

20 

24 

67 

TABLE  VII 

Strontium  Caseinate.     Neutral  to  Phenolphthalein.     80 X10~5  Equiva- 
lents Sr(OH)2  per  gram.    Temperature  30  degrees 

A  =32. 65        5  =  4930 


m  X  10* 

(experimental) 

m  X  104 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

224 

226 

61 

168 

167 

66 

112 

106 

74 

84 

86 

77 

56 

58 

.83 

42 

47 

85 

TABLE  VIII* 

Barium  Caseinate.     Neutral  to  Phenolphthalein.     80X10"5  Equiva- 
lents Ba(OH)2  per  gram.    Temperature  30  degrees 

A  =23. 73        5  =  23,400 


m  X  10* 
(experimental) 

m  X  10* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

320 

326 

24 

160 

147 

33 

80      • 

76 

42 

40 

41 

54 

20 

22 

64 

*  For  data  used  in  Tables  V,  VI,  VII  and  VIII,  Cf.  T.  Brailsford  Robertson  (15)  (17). 


DILUTION 


225 


TABLE  IX* 

Potassium  Caseinate.    90X10~6  Equivalents  KOH  per  gram.    Tem- 
perature 30  degrees 
A  =  11.59        B  =  1739 


mXlO* 
(experimental) 

mXlO* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

180 

180 

83 

135 

135 

87 

90 

90 

90 

45 

46 

96 

23 

25 

96 

*  The  conductivity  of  the  trace  of  free  KOH  was  in  no  case  more  than  2  per  cent  of  the  observed 
conductivity;  the  observed  conductivities  were  not  corrected  for  this  (18). 


Potassium  Caseinate. 


TABLE  X* 
100X10-6  Equivalents  KOH  per  gram. 

perature  30  degrees 
A  =  9.57        B  =  1242 


Tern- 


mXlO4 
(experimental) 

TOX10* 

(calculated) 

Degree  of  dissocia- 
tion, per  cent 

300 

300 

76 

200 

201 

82 

150 

149 

85 

100 

100 

89 

50 

53 

93 

*  Calculated  for  solutions  containing  small  amounts  of  KC1  according  to  the  method  described 
in  the  previous  chapter  (section  1)  and,  therefore,  corrected  for  the  conductivity  of  the  free 
KOH  as  determined  by  gas-chain  measurements.  T.  Brailsford  Robertson  (14). 


TABLE  XI* 

Potassium    Paranucleinate.     Neutral    to    Phenolphthalein.    36X10"5 

Equivalents  KOH  per  gram.     Temperature  25  degrees 

A  =  12.38        B  =  3784 


mXlO4 
(experimental) 

mX10< 

(calculated) 

Degree  of  dissocia- 
tion, per  cent 

92 

93 

85 

46 

46 

93 

23 

23 

96 

12 

13 

100 

6 

7 

100 

*  T.  Brailsford  Robertson  (12). 


226 


ELECTROCHEMISTRY 


TABLE  XII* 

Sodium  Serum-globulinate.     Neutral  to  Litmus.    9.75X10~5  Equiv- 
alents NaOH  per  gram.    Temperature   18  degrees 

A  =  17.65        B  =  36,200 


m 
(experimental) 

m 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

657 

657 

66 

329 

302 

78 

164 

155 

87 

82 

83 

92 

41 

44 

96 

21 

29 

97 

*  Calculated  from  experimental  data  obtained  by  W.  B.  Hardy  (2). 

The  constants  determined  by  least  squares  (A  =  22.23;  B  =  17,391)  do  not  give,  for  the  smaller 
values  of  m,  so  good  an  agreement  as  the  above.  From  the  evidence  afforded  by  the  numerous 
data  set  forth  in  these  tables  there  is  strong  presumptive  reason  for  believing  that,  notwithstanding 
this  lack  of  agreement,  the  dilution-law  holds  good  in  this  case  also,  and  that  experimental  errors 
are  responsible  for  the  deviations.  A  very  probable  source  of  error  in  these  determinations  is,  as 
Hardy  points  out,  the  conductivity  of  the  undissolved  suspension  of  globulin,  a  quantity  of  very 
uncertain  magnitude  and  meaning.  This  conductivity  was  very  appreciable  with  the  globulin 
employed  by  Hardy  (precipitated  by  acetic  acid),  while  it  was  unappreciable  with  the  globulin 
employed  in  my  experiments  (precipitated  by  CO2).  Since  the  method  of  least  squares  fails  to 
yield  constants  which  satisfy  the  experimental  results,  I  have  taken  A  for  this  salt  to  be  the  same 
as  A  for  the  salt  containing  18  X  10~5  equivalents  NaOH  per  gram.  B  is  estimated  from  the  first 
observation.  It  is  evident  that  the  order  of  agreement  between  the  calculated  and  observed 
values  is  tolerably  good. 


TABLE  XIII* 

Sodium   Serum-globulinate.     Neutral    to    Phenolphthalein.     18X10"8 
Equivalents  NaOH  per  gram.     Temperature  18  degrees 

A  =  17.65        B  =  11,500 


mX10« 
(experimental) 

mX10< 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

313 

310 

59 

156 

156 

71 

78 

77 

81 

39 

38 

88 

10 

10 

95 

*  Calculated  from  the  experimental  data  of  W.  B.  Hardy  (2). 


DILUTION 


227 


TABLE  XIV* 

Serum-globulin   Chloride.     9.318X10~5   Equivalents   HC1   per   gram. 
Temperature  18  degrees 

A  =  7.42        B  =  36,964 


m 
(experimental) 

m 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

933 

926 

33 

461 

484 

42 

231 

219 

55 

115 

105 

68 

58 

54 

78 

29 

30 

83 

*  Calculated  from  the  experimental  data  of  W.  B.  Hardy  (2). 


TABLE  XV 

Potassium  Serum-globulinate.     Neutral  to  Phenolphthalein.     20X10"5 
Equivalents  KOH  per  gram.     Temperature  30  degrees 

A  =  19.62        B  =  51,900 


m  X  105 
(experimental) 

TO  X  106 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

296 

297 

77 

148 

147 

85 

'    74 

70 

92 

37 

37 

95 

18 

18 

98 

TABLE  XVI* 

Calcium  Serum-globulinate.     Neutral  to   Phenolphthalein.     20X10"5 
Equivalents    Ca(OH)2   per    gram.     Temperature    30    degrees 

A  =  43.25        B  =  502,000 


TO  X  10* 
(experimental) 

TOX105 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

296 

296 

66 

148 

146 

77 

74 

76 

85 

37 

43 

91 

18 

18 

96 

*  Fourth  observation  omitted  in  computing  the  constants. 


228 


ELECTROCHEMISTRY 


TABLE  XVII 

Strontium  Serum-globulinate.     Neutral  to  Phenolphthalein.    20X10"5 
Equivalents  Sr(OH)2  per  gram.     Temperature  30  degrees 

A  =  32.85        B  =  628,000 


mXlQs 
(experimental) 

TO  X  10* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

74 

74 

76 

37 

36 

82 

18 

19 

87 

TABLE  XVIII* 

Barium   Serum-globulinate.     Neutral    to    Phenolphthalein.    20X10"6 
Equivalents  Ba(OH)2  per  gram.    Temperature  30  degrees 

A  =  42.61        B  =  369,000 


TO  X  105 
(experimental) 

TO  X  105 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

296 

297 

70 

140 

142 

81 

74 

78 

87 

37 

41 

93 

18 

18 

94 

*  For  the  data  utilized  in  Tables  XV,  XVI,  XVII,  XVIII,  Cf.  T.  Brailsford  Robertson  (17). 


TABLE  XIX* 

Ovomucoid  Chloride.    45X10"5  Equivalents  HC1  per   gram.    Tem- 
perature 30  degrees 

A  =  5.103        B  =  525 


TO  X  10* 

(experimental) 

m  X10* 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

180 

181 

78 

90 

89 

87 

45 

44 

93 

23       - 

22 

96 

11 

11 

97 

*  Free  HC1  =  less  than  0.5  X  10~4  for  the  1  per  cent  solution;  observed  conductivity  not  cor- 
rected  for  this.    Data  utilized  in  this  and  the  following  table  from  T.  Brailsford  Robertson  (16). 


DILUTION 


229 


Ovomucoid  Sulphate. 


TABLE  XX 

45X10"5  Equivalents  H2S04  per  gram. 
perature  30  degrees 

A  =  5.488        B  =  986 


Tem- 


m X  104 

(experimental) 

mXlO4 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

180 

180 

71 

90 

89 

81 

45 

45 

89 

23 

23 

96 

11 

12 

96 

TABLE  XXI 

Protamin  (Salmin)  Sulphate.    424X10~6  Equivalents  H2S04  per  gram. 
Temperature  30  degrees 

A  =  9.09        B  =  6408 


mXlO* 
(experimental) 

mX10< 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

105.3 

105.7 

65 

84.2 

83.4 

69 

52.6 

52.7 

76 

26.3 

26.7 

85 

13.2 

13.7 

94 

6.6 

7.1 

100 

3.3 

3.5 

100 

TABLE  XXII 

Protamin  (Salmin)  Chloride.    424X10~5  Equivalents  HC1  per  gram. 
Temperature  30  degrees 

A  =  6.92          B  =  1781 


mXlO4 
(experimental) 

mXlO4 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

55.8 

55.9 

85 

27.9 

27.6 

92 

13.9 

14.1 

95 

7.0 

7.1 

97 

3.5 

3.6 

100 

230 


ELECTROCHEMISTRY 


It  will  be  observed  that  as  a  rule  the  agreement  between  the 
calculated  and  the  observed  values  of  m  is  excellent  and  we  may 
therefore  conclude  that  equation  (ii)  (or  (iii))  represents  the 
dependence  of  conductivity  upon  dilution,  for  the  salts  of  the 
proteins,  with  very  satisfactory  fidelity.  Recollecting  the  mode 
of  derivation  of  equation  (ii)  this  fact  might  be  held  to  prove  that 
the  protein  salts,  in  all  of  the  solutions  investigated,  dissociate 
into  two  ions.  This  deduction  is  not  altogether  a  safe  one,  how- 
ever. The  generalized  form  of  equation  (iii)  for  electrolytes  which 
dissociate  into  n  ions  is:* 

m  =  Ax  +  Bxn  (iv) 

and,  provided  the  departure  of  equation  (iv)  from  the  linear 
form  is  small,  the  factor  Bxn  may  be  represented,  within  the 
experimental  error,  by  Bx2  even  if  n  be  in  reality  a  higher  ex- 
ponent than  2,  while  vice  versa,  Bx2  may  be  approximated  by 
Bxn,  where  n  is  a  higher  exponent  than  2.  For  example,  in  the 
following  table  the  values  of  m  for  calcium  caseinate  solutions 
(Cf.  Table  VI),  calculated  from  formula  (iii)  and  from  the  formula 

m  =  A&  +  £iz3,  (v) 

are  compared,  f 

TABLE  XXIII 


rnXlO* 
(experimental) 

mX  103 
(calculated  from 
formula  (iii)) 

mX10» 
(calculated  from 
formula  (v)) 

32 
24 
16 
12 

8 
6 
4 
2 

32 
24 
15 

12 
8 
7 
5 
2 

33 
23 
14 
11 

8 
7 
5 
3 

SA=  +1 

SA  =  ±0 

*  If  the  electrolyte  dissociates  partly  into  2,  partly  into  3,  partly  into  4,  etc., 
ions  the  equation  becomes: 

m  =  Ax  +  Bx*  +  Cz3  •  •  •  +  Nxn. 

t  Sutherland  (22)  (23),  urging  the  applicability  of  his  theory  of  ionization 
to  the  salts  of  proteins,  has  pointed  out  that  the  results  of  Hardy  (cited  above, 
Table  XIV)  obtained  with  HCl-globulin  containing  9.32  X10~6  equivalents- of 
acid  per  gram  of  protein  may  be  expressed  by  the  equation: 

-  =  0.00026  +  0.009  tr*, 
M 

in  which  /t  is  the  molecular  conductivity  of  the  salt,  calculated  in  terms  of 
HCl-concentrations,  and  v  is  the  volume  of  solution  containing  one  equivalent 
of  Cl. 


DILUTION  231 

There  is  little  to  choose  between  the  two  formulae,  although, 
on  the  whole,  the  agreement  between  the  values  calculated  from 
formula  (iii)  and  the  experimental  values  is  somewhat  closer 
than  that  between  the  values  calculated  from  formula  (v)  and 
the  experimental  values.  As  we  shall  see,  however,  the  internal 
evidence  which  is  afforded  by  the  numerical  values  of  the  con- 
stants demonstrates  that  the  true  relationship  between  dilution 
and  conductivity  for  solutions  of  the  salts  which  proteins  form 
with  bases  is  that  which  is  expressed  by  equation  (iii);  namely 
that  which  is  characteristic  for  an  electrolyte  which  yields  two 
ions  per  molecule. 

As  regards  the  salts  which  proteins  form  with  acids,  the  binary 
formula  expressed  by  equation  (iii)  yields  demonstrably  better 
agreement  with  the  experimental  data  obtained  with  salmin 
sulphate  and  chloride  than  the  ternary  formula  expressed  by 
equation  (v).  The  salts  which  protamins  form  with  acids  would 
therefore  appear  to  yield  only  two  ions,  irrespective  of  the  valency 
of  the  acid  anion  with  which  the  protein  is  combined.  On  the 
other  hand,  the  relationship  of  conductivity  to  dilution  which  is 
displayed  by  the  salts  which  ovomucoid  forms  with  hydrochloric 
acid  is  more  adequately  expressed  by  the  ternary  formula. 

It  will  be  recollected  that  the  constants  A  and  B  in  equation 
(iii)  represent,  respectively,  the  quantities 

1.037  X  10-2  1.075  X  104 


where  u  -\-  v  is  the  sum  of  the  equivalent  migration  velocities  of 
the  ions  in  centimeters  per  second  under  a  potential  gradient 
of  1  volt  per  centimeter,  p  is  the  number  of  equivalents  of  the 
protein  salt  which  is  yielded  by  the  neutralization  of  one  equiva- 
lent of  inorganic  base  or  acid,  and  K  is  the  dissociation  constant 

of  the  salt.     It  will  be  observed  that  ^  =  —  •     We  can,  therefore, 

K        p 

from  the  data  enumerated  in  the  above  tables,  compute  p  (u  +  v) 
jr 

and  —  for  each  of  the  salts  investigated.  The  values  of  these 
constants  follow. 


232 


ELECTROCHEMISTRY 


TABLE  XXIV. 


CASEINATES  OF  INORGANIC  BASES 

Caseinates  of  monacid  bases 


Nature  of  the  base  and  proportion  of  base 
to  1  gram  protein 

Temp., 
degrees 

p(u+v) 

K 
P 

50X10"5  NaOH  per  gram  

25 

53X10~5 

40X1Q-3 

80X10"5  NaOH  per  gram  

25 

66X10~5 

50X10-3 

50XKT5  NH4OH  per  gram  

25 

63X10~5 

44X10-3 

80X10~5  NH4OH  per  gram 

25 

82X10"5 

40X1Q-3 

80X10"5  KOH  per  gram   . 

30 

84X10"5 

91  X  10"3 

90X10"5  KOH  per  gram.  .  . 

30 

89X10~5 

77X10"3 

100  X  10~5  KOH  per  gram 

30 

108X10"5 

74X10"3 

Caseinates  of  diacid  bases 


80X10"5  Ca(OH)  per  gram. 

30 

37X10~5 

2  9X10~3 

80X10"5  Sr(OH)2  per  gram 

30 

32X10~5 

21  6X10"3 

80X10~5  Ba(OH)2  per  gram  

30 

44X10-5 

2.4X10-3 

TABLE  XXV. 


SERUM  GLOBULINATES  OF  INORGANIC  BASES 

Globulinates  of  monacid  bases 


Nature  of  the  base  and  the  proportion  of 
base  to  1  gram  protein 

Temp., 
degrees 

p(u  +  v) 

K 

P 

9.75X10~5  NaOH  per  gram  
18.0X10~5  NaOH  per  gram  
20.  OX10~5  NaOH  per  gram  

18 
18 
30 

59X10-5 
59X10-6 
53X10~5 

8.6X10-3 
27.0X10-3 
7  4X10~3 

Globulinates  of  diacid  bases 


20X10~5  Ca(OH)2  per  gram  

30 

24X10-5 

3.7X10-3 

20X10-5  Sr(OH)2  per  gram  
20X10~5  Ba(OH)2  per  gram  

30 
30 

32X10-5 
24X10~5 

1.7X10-3 
4.9X10-3 

TABLE  XXVI.    COMPOUNDS    OF    PROTEINS    WITH    ACIDS 

Monobasic  acids 


Nature  of  the  acid  and  protein  and  the  pro- 
portion of  acid  to  1  gram  protein 

Temp., 
degrees 

p(u-HO 

K 
P 

9.318X10~5  HC1  per   gram   serum 
globulin                 

18 

140X10"5 

1.5X10-3 

45X10~5  HC1  per  gram  ovomucoid.  . 
442X10~5  HC1  per  gram  salmin  

30 
30 

203X10-5 
150X10-5 

50     X10-3 
26.9X10-3 

Dibasic  acids 


45X10-5  H2SO4 
coid 

per  gram  ovomu- 

30 

189X10~5 

31     X  10-3 

442X10-5H2SO4 

per  gram  salmin  .  . 

30 

114X10-5 

12.9X10-3 

For  potassium  paranucleinate,  containing  36  X  10~5  equivalents  of  KOH  per  gram,  the  value 

of  p(u  + 1>)  is  84  X  10-6  and  that  of  -  is  41  X  10~8. 
P 


DILUTION  233 

On  surveying  these  tables  several  striking  facts  are  immedi- 
ately revealed.  In  the  first  place  the  ratio  of  the  value  of  p  (u  +  v) 
for  monacid  bases  to  its  value  for  diacid  bases  is,  in  every  case 
(Cf.  Tables  XXIV  and  XXV)  exceedingly  close  to  the  ratio  2:1. 
This  may  be  interpreted  in  either  of  two  ways;  either  the  equiva- 
lent velocity  of  the  protein  ions  which  are  yielded  by  the  salts 
of  monacid  bases  is  twice  as  great  as  that  of  the  ions  which  are 
yielded  by  the  salts  of  diacid  bases,  or  else  one  equivalent  of  a 
monacid  base  gives  rise  to  twice  as  many  equivalents  of  protein 
salt  as  one  equivalent  of  a  diacid  base.  We  have  seen  that  the 
protein  salts  dissociate  into  two  heavy  protein  ions  and,  as  Bredig 
has  shown  (1),  the  equivalent  velocities  of  heavy  ions  approach 
a  constant  minimum.  The  former  of  the  above  alternatives  may 
therefore  be  dismissed,  and  we  must  conclude  that  p,  the  number 
of  equivalents  of  protein  salts  to  which  one  equivalent  of  base 
gives  rise,  is  twice  as  great  for  the  monacid  as  for  the  diacid  bases. 

Reverting  to  the  hypothesis  developed  in  the  preceding  chap- 
ters, it  appears  that  the  monacid  bases  form  salts  with  proteins 
in  the  following  way : 

H 

++  I 

-COH.N-  +  NaOH  =  -  CONa  +  ^N- 

I 
OH 

from  which  it  is  evident  that  for  the  salts  of  the  monacid  bases 
p  must  be  2,  i.e.,  one  equivalent  of  a  monacid  base,  when  united 
with  protein,  yields  2  equivalents  of  the  protein  salt.  We  must 
conclude,  then,  that  one  equivalent  of  diacid  base  gives  rise  to 
only  one  equivalent  of  protein  salt.  The  most  natural  assumption 
regarding  the  mode  of  combination  of  the  proteins  with  diacid 
bases,  pursuing  the  above  hypothesis,  would  be  to  suppose  that 
it  takes  place  in  accordance  with  the  equation: 

H 

-CO++  I 

2-COH.N-  +Ca(OH)2=  ;)Ca  +  2^N- 

-C0++x  I 

OH 

upon  which  supposition  each  equivalent  of  the  diacid  base  would 
give  rise  to  just  the  same  number  of  equivalents  of  protein  salt 
as  an  equivalent  of  a  monacid  base,  namely  2.  This  is  evidently 


234  ELECTROCHEMISTRY 

not  the  case,  and  we  must  assume  that  two  of  the  positive  valen- 
cies of  the  cation  are  neutralized  and  at  the  same  time  that  two 
of  the  negative  valencies  which  the  anions  supply  are  also  neu- 
tralized. This  immediately  suggests  that  one  of  the  two  anions 
neutralizes  two  of  the  valencies  of  the  cation  and  that  the  true 
constitution  of  the  cation  is  either 


-CO++     v  -CO+,       H 

)Ca  I    \  I 

-CO.N-  or  Ca      >N- 

/\  I    /  \ 

H     OH  -CO+/     OH 

or  either  of  these  formulae  less  the  elements  of  water  which  are 
attached  to  the  nitrogen  atom.  If  this  be  so  then  we  must  con- 
clude that  each  molecule,  of  a  diacid  base  gives  rise  to  exactly 
the  same  number  of  ions  (apart  from  the  question  of  the  degree 
of  dissociation  of  the  salt)  and  equivalents  of  protein  salt  as  a 
molecule  of  a  monacid  base,  and  that  the  molecule  of  a  protein 
salt  of  a  diacid  base  must  be  twice  as  heavy  as  a  molecule  of  a  protein 
salt  of  a  monacid  base.  In  this  connection  the  following  differ- 
ences between  the  physical  behavior  of  solutions  of  the  caseinates 
of  the  alkalies  and  those  of  the  caseinates  of  the  alkaline  earths 
may  be  recalled  to  the  reader:  The  aqueous  solutions  of  the 
"  neutral"  or  "  basic"  caseinates  of  the  alkalies  and  ammonium 
are  clear,  do  not  (except  lithium  caseinate)  show  any  increase  in 
turbidity  on  warming,  and  are  not  precipitated  by  the  addition 
of  finely  divided  insoluble  substances  or  by  passing  through  a 
clay  filter,  while  the  aqueous  solutions  of  the  caseinates  of  the 
alkaline  earths  are  opalescent,  show  a  marked  turbidity  on  heat- 
ing their  solutions  to  35-45°  C.,  which  disappears  on  cooling, 
and  are  precipitated  from  their  solutions  by  the  addition  of 
finely  divided  insoluble  substances  or  by  passage  through  a  clay 
filter  (5).  Equally  concentrated  solutions  of  the  hydroxides  of 
the  alkalies  and  ammonium  dissolve  casein  at  approximately 
the  same  rate.  Solutions  of  the  hydroxides  of  the  alkaline  earths 
dissolve  casein  much  more  slowly  (9)  (13). 

The  salts  which  casein  forms  with  monacid  bases  at  "satu- 
ration" of  the  base  with  casein,  that  is,  when  the  proportion  of 
base  to  casein  is  11.4  X  10~5  equivalents  per  gram,  are  soluble 
in  water,  while  the  corresponding  salts  which  casein  forms  with 


DILUTION  235 

diacid  bases  are  insoluble  in  water  (11)  (25).  All  of  these  differ- 
ences, it  will  be  observed,  suggest  more  or  less  strongly  that  the 
molecules  of  the  caseinates  of  the  alkaline  earths  are  of  greater 
size  and  more  ponderous  than  those  of  the  casemates  of  the 
alkalies. 

Assuming,  therefore,  that  p  =  2  f  or  the  salts  of  the  alkalies 
and  1  for  the  salts  of  the  alkaline  earths  we  find  that  the  numer- 
ical values  of  p  (u  -\-  v)  in  Tables  XXIV  and  XXV  accord  very 
well  with  our  knowledge  of  the  mobilities  of  heavy  ions.  The 
average  value  of  u  +  v  at  30  degrees  for  potassium  caseinate 
containing  80  X  10~5  equivalents  of  KOH  per  gram  is  42  X  10~5 
cm.  per  sec.,  while  for  the  salts  of  the  alkaline  earths  at  the  same 
temperature  the  average  value  of  u  -\-  v  is  38  X  10~5.  Assuming 
the  migration  velocities  of  the  anions  and  cations  to  be  equal 
(since,  as  we  have  seen,  their  masses  are  nearly  equal)  the  equiv- 
alent velocity  of  a  protein  ion  at  30  degrees  would  appear  to  be 
about  20  X  10~5  cm.  per  sec.  under  a  potential  gradient  of  1  volt 
per  centimeter.  Now  Bredig  (1)  has  shown  that  the  equivalent 
velocities  of  heavy  ions  tend  to  attain,  with  increasing  weight, 
a  constant  minimum  value  of  about  15  X  10~5  cm.  per  sec.  at 
15  degrees.  At  30  degrees  this  velocity  would  be  increased  by 
about  30  per  cent,  i.e.,  would  become  20  X  10~5  cm.  per  sec. 

Referring  to  Table  XXVI  it  is  evident  that  p  (u  +  0)  is  much 
larger  for  the  salts  which  proteins  form  with  acids  than  it  is 
for  the  salts  which  they  form  with  bases.  It  is  about  twice  as 
large,  in  the  case  of  serum  globulin,  as  it  is  for  the  salts  of  diacid 
bases.  We  can  hardly  assume  that  the  migration-velocity  of 
protein  ions  is  twice  as  great  in  acid  as  in  alkaline  solution  and 
we  must  therefore  adopt  the  alternative  assumption  that  p  is 
twice  as  great  in  acid  as  in  alkaline  solutions.  This  might  arise 
by  the  splitting  of  double  —  COH.N—  bonds,  thus: 

H    Cl 
-COH.N,  -COH 


-  COH.N  -COH 

/  \ 
H    OH 

which  indicates  that,   as  Kossel  has  suggested   (3)   the  active 
agents  in  bringing  about  the  neutralization  of  acids  by  proteins 


236  ELECTROCHEMISTRY 

are  the  diamino  radicals  (hexone  bases)  which  are  contained  in 
the  protein  molecule.  As  we  shall  see  in  the  succeeding  section 
of  this  chapter,  the  neutralization  of  bases  by  the  proteins  is 
analogously  accomplished,  at  any  rate  for  the  greater  part,  by 
the  dicarboxylic  radicals  which  they  contain;  only  the  evidence 
which  we  have  so  far  discussed  does  not  reveal  this  fact  because 
salts  of  the  type 

H     OH 


.COK 

R'    ++  + 

XCOH 

/  \ 
H     OH 

do  not  appear  to  exist,  but  only  salts  of  the  type 

H     OH 


COK 

/  V 

H     OH 

in  which  the  ratio  of  the  valencies  of  the  cation  to  the  number 
of  equivalents  of  base  included  in  the  molecule  of  the  protein 
salt  is  2;  the  same  as  it  would  be  for  a  salt  formed  by  the  open- 
ing up  of  a  single  —  COH.N—  bond. 

The  results  obtained  with  solutions  of  ovomucoid  in  dilute 
acids  are  complicated  by  the  possible  presence  of  free  ovomucoid 
which  is  itself  ionized  and  therefore  contributes  to  the  conduc- 
tivity of  these  solutions.  It  is  evident,  however  (Cf.  Tables  XXI 
and  XXII),  that  the  conductivity  of  a  solution  of  ovomucoid 
sulphate  is  practically  identical  with  that  of  a  solution  of  ovo- 
mucoid chloride  containing  the  same  equivalent  concentration 
(45  X  10~5  equivalents  per  gram)  of  neutralized  HC1.  Hence  it 
follows  that  in  the  formation  of  these  salts  each  equivalent  of 
a  dibasic  acid  must  give  rise  to  the  same  number  of  equivalents 
of  protein  salt  that  an  equivalent  of  a  monobasic  acid  produces. 
This  conclusion  also  applies  to  the  salts  which  the  protamin, 
salmin,  forms  with  acids  (19). 

2.  The  Depression  of  the  Freezing-point  of  Water  which  is 
Caused  by  Dissolved  Protein  Salts,  and  the  Stoichiometry  of 


DEPRESSION  OF  THE  FREEZING-POINT  237 

Protein  Salts.  —  We  have  hitherto  found  it  convenient  in  dis- 
cussing the  mode  of  formation  of  the  salts  which  the  proteins 
form  with  bases,  only  to  consider  the  consequences  which  arise 
out  of  the  opening  up  of  a  single  —  COH.N—  bond,  without 
involving  a  consideration  of  the  question  whether  this  —  COH.N  — 
bond  is  supplied  by  a  dicarboxylic  acid  group  (such  as  glutamic 
acid),  in  which  case  two  such  bonds  must  be  opened  up  before 
actual  electrolytic  dissociation  can  occur,  or  whether  it  is  supplied 
by  a  mono-carboxylic  acid  group  (such  as  glycocoll)  in  which 
case  dissociation  could  occur  directly  one  such  group  was  opened 
up.  We  have  been  able  to  do  this  because,  in  the  formation  of 
salts  with  bases,  all  of  the  —COH.N—  groups  which  are  opened 
up  suffer  the  introduction  of  the  basic  radical  and  the  equiva- 
lence between  the  protein  salt  and  the  base  which  gives  rise  to 
it  is  the  same  as  that  which  would  subsist  were  the  salt  formed 
by  the  opening  up  of  only  one  —COH.N—  group.  All  of  the 
preceding  considerations  and  theoretical  deductions,  therefore, 
remain  equally  valid  whether  the  —COH.N—  groups  under 
consideration  in  each  separate  case  are  derived  from  a  dicarboxylic 
acid  radical  or  from  a  mono-carboxylic  acid  radical.  Only,  so 
far  as  our  experimental  knowledge  goes,  when  we  investigate 
the  freezing-point  depression  (i.e.,  the  osmotic  pressure)  of  solu- 
tions of  protein  salts  do  we  encounter  facts  which  are  definitely 
irreconcilable  with  the  view  that  the  protein  salts  with  bases 
are  formed  through  the  agency  of  mono-carboxylic  acid  radicals 
and  which  point  unmistakably  to  the  dicarboxylic  add  radicals 
as  the  active  agents  in  accomplishing  these  unions. 

It  has  been  shown  by  Robertson  and  Burnett  (20)  that  whether 
the  caseinate  formed  is  neutral  to  litmus  and  contains  50  X  10~5 
equivalents  of  base  per  gram,  or  neutral  to  phenolphthalein  and 
containing  80  X  10~5  equivalents  of  base  per  gram,  the  freezing- 
point  depression  which  is  brought  about  by  the  dissolved  protein  salt 
bears  the  same  proportion  to  the  concentration  of  the  neutralized  base. 

The  significance  of  this  observation  has  been  alluded  to  in 
the  latter  part  of  Chap.  I.  If  we  prepare  a  number  of  solutions 
all  containing  the  same  amount  of  a  base  and  add  to  these  vary- 
ing amounts  of  a  polybasic  acid  R(COOH)n,  such  that  in  the 
first  solution  only  one,  in  the  second  two,  etc.,  —  COOH  groups 
are  neutralized,  then,  if  all  these  salts  are  highly  dissociated, 
calling  the  osmotic  pressure  of  the  first  1,  that  of  the  second 


238  ELECTROCHEMISTRY 

will  be  f,  that  of  the  third  f,  etc.  But  if,  to  solutions  which 
contain  the  same  amount  of  a  base  we  add  varying  amounts 
of  casein,  the  osmotic  pressures  of  all  of  these  solutions,  as  indi- 
cated by  their  freezing-points  are  the  same.  Evidently,  therefore, 
a  given  quantity  of  base  always  gives  rise  to  the  same  number 
of  protein  ions,  whether  the  base  is  combined  with  more  or  with 
less  protein.  This  obviously  corresponds  with  the  view  that 
each  equivalent  of  base  opens  up  a  given  number  of  —  COH.N  — 
groups,  and  not  at  all  with  the  view  that  it  neutralizes  terminal 
-COOH  groups. 

The  experimental  data  which  were  obtained  by  Robertson 
and  Burnett  are  enumerated  in  Tables  I  and  II  in  Chap.  XIII 
(p.  333).  On  referring  to  the  results  which  are  therein  cited 
it  will  be  observed  that  the  depression  of  the  freezing-point  which 
is  brought  about  by  the  dissolved  caseinate  of  the  alkalies  is  in 
every  case  very  nearly  that  which  would  be  observed  in  a  solu- 
tion of  the  same  molecular  concentration  as  that  of  the  alkali 
which  is  neutralized  by  the  casein.  Now  these  solutions  are 
quite  extensively  (over  80  per  cent,  Cf.  Tables,  section  1)  dissoci- 
ated and  it  therefore  follows  that  each  molecule  of  alkali  gives  rise 
to  one  ion  of  caseinate.  That  the  same  is  true  for  the  alkaline 
earths  is  readily  seen  when  allowance  is  made  for  their  compara- 
tively slight  degree  of  dissociation  (Cf.  Tables  VI,  VII  and  VIII). 
Now  in  the  formation  of  a  salt  by  the  splitting  of  a  single 
—  COH.N—  bond  each  molecule  of  neutralized  base  would  give 
rise  to  two  ions  of  caseinate,  while  in  the  formation  of  a  salt  by 
the  splitting  of  a  double  —COH.N—  bond  one  molecule  of 
neutralized  base  might  give  rise  to  only  one  ion  of  caseinate. 
This  will  be  clear  from  the  following  equations: 

H 

++  I 

-COH.N-  +  NaOH  =  -CONa+  ^N- 

I 
OH 

H  OH 
COH.N.  CONa 


COH.N  CONa 

/  \ 
H    OH 


DEPRESSION  OF  THE  FREEZING-POINT  239 

Evidently,  therefore,  it  is  the  latter  alternative  which  repre- 
sents the  true  mode  of  formation  of  these  salts,  and  we  must 
conclude  that  in  the  neutralization  of  bases  the  dicarboxylic 
radicals  play  a  leading  part,  just  as,  in  the  neutralization  of 
acids,  the  diamino  radicals  play  a  leading  part. 

We  have  seen  that  in  the  formation  of  ovomucoid  chloride, 
containing  45  X  10~5  equivalents  of  acid  per  gram,  only  one  of 
the  —  COH.N—  bonds  which  is  opened  up  suffers  the  introduction 
of  an  HC1  molecule.  In  this  case,  therefore,  if  the  above  reason- 
ing has  been  correct,  the  freezing-point  depression  due  to  the  dis- 
solved salt  should  be  either  twice  or  three  times  that  of  a  solution 
of  the  molecular  concentration  of  the  neutralized  acid  according 
as  to  whether  the  formation  of  the  salt  is  represented  by  the 
equation: 

H   Cl 

++        \  / 

,  COH.N,  /COH 

R'  ^R  +  HC1  +  H20  =  R'     ++ 

x  COH.N /  XCOH 

/  \ 
H  OH 
or  by  the  equation: 

H    C 
\  / 
R-COH.N.  ^N.x 

^  R  +  HC1  +  H20  =  2  R.COH+++         ^  R 
R-COH.NX  ^Nx 

/  \ 
H  OH 

that  is,  according  as  to  whether  the  diamino  acid  radicals  in  the 
ovomucoid  molecule  are  directly  united  with  both  carboxyls  of 
dicarboxylic  acid  radicals,  or  with  only  one  or  with  monocar- 
boxylic  acid  radicals.  The  experimental  fact  is  that  the  freezing- 
point-depression  of  a  0.018  m.  solution  of  HC1  containing  4  per 
cent  of  ovomucoid  (1  gram  per  45  X  10~5  equivalents)  is  0.09  ± 
0.005  degree,  corresponding  to  a  0.055  m.  solution,  or  almost 
exactly  three  times  the  molecular  concentration  of  the  neutralized 
acid. 

Hence  we  may  conclude  that  the  formation  of  this  salt  is  repre- 
sented by  the  latter  of  the  above  two  equations  and  that  in  ovo- 
mucoid the  diamino  radicals  are  not  directly  attached  to  both  carboxyls 
of  dicarboxylic  radicals. 


240 


ELECTROCHEMISTRY 


Continuing  the  addition  of  acid  (HC1)  to  the  above  solution 
of  ovomucoid  a  remarkable  phenomenon  is  observed,  namely, 
that  doubling  the  amount  of  acid  in  the  solution  does  not  appreciably 
alter  its  freezing-point  *  and,  consequently  does  not  alter  the  total 
number  of  ions  per  cubic  centimeter  of  the  solution.  Evidently, 
upon  further  addition  of  acid,  the  remaining  nitrogen  atom  of  the 
diamino  radical  becomes  neutralized  and  the  ion 

H    Cl 
\  / 


\ 


R 


/  \ 

is  formed.  H    C1 

The  form  of  the  dilution-law  which  we  should  apply  to  these 
solutions  is  therefore  (Cf.  equation  (v))  : 


m 


•  1.037  X  10~2 


1.115  X  1Q-6 


x*. 


P  (u  +  v) 

Applying  this  equation  to  the  results  enumerated  in  Table 
XIX  we  obtain         m  =  5.68  x  +  117,000  x3. 

In  the  accompanying  table  the  experimental  and  calculated  values 
of  m  are  compared.  In  the  third  column  is  given  the  degree  of 
dissociation  of  the  salt,  estimated  as  the  ratio  of  the  calculated 
value  of  5.63  x  to  the  calculated  value  of  m. 

TABLE  XXVII.    OVOMUCOID  CHLORIDE 
(          (45X10~5  equivalents  HC1  per  gram) 


mXlO* 
(experimental) 

mXlO4 
(calculated) 

Degree  of  dissocia- 
tion, per  cent 

180 

180 

86 

90 

89 

96 

45 

46 

98 

23 

24 

100 

11 

12 

100 

The  same  equation  applies  so  nearly  to  the  experimental  results 
obtained  with  ovomucoid  sulphate  (Table  XX)  that  it  is  not 
necessary  to  recompute  the  constants  for  this  salt. 

*  The  freezing-point-depression  due  to  free  ovomucoid  is  only  one-third  of 
that  of  the  above  solution,  namely,  0.03  ±  0.005  degree  for  a  4  per  cent 
solution. 


DEPRESSION  OF  THE  FREEZING-POINT  241 

1  037  X  10~2 

From  the  value  of   -  —  -,  —  ;  —  r—  for  the  ovomucoid  chloride 
P  (u  +  v) 

we  obtain:  P(u  +  v)  =  134  X  lO"5, 

whence,  if  p  =  4,  u  +  v  =  46  X  10~5,  which  agrees  very  well 
with  the  values  obtained  with  other  protein  salts  for  this  constant 
(Tables  XXIV  and  XXV;  Cf.  also  p.  235). 

A  one-half  per  cent  solution  of  salmin  hydrochloride  freezes 
at  a  temperature  of  0.04  degrees  lower  than  distilled  water. 
This  corresponds  to  the  freezing-point  depression  of  a  solution 

in  which  the  molecular  +  ionic  concentration  is   -^  .     Now  the 

46 

equivalent  concentration  of  HC1  neutralized  by  salmin  in  a  J 

AM 

per  cent  solution  of  salmin  hydrochloride  is  j=.     Each  molecule 

of  combined  hydrochloric  acid  yields,  therefore,  one  molecule 
or  one  ion  of  salmin  hydrochloride.  Salmin  hydrochloride  in 
|  per  cent  solution  is  very  highly  ionized,  since  dilution  only 
increases  its  equivalent  conductivity  to  a  small  extent  and  we 
must  infer,  therefore,  since  the  salt  exists  in  solution  largely  in 
the  form  of  ions,  that  each  molecule  of  combined  acid  yields 
one  ion  of  the  protein  salt.  One  molecule  of  salmin  hydrochloride 
contains  at  least  four  molecules  of  combined  hydrochloric  acid 
(3)  (24),  hence  one  molecule  of  salmin  hydrochloride  must  yield 
four  ions  or  a  multiple  thereof.  From  the  value  of  the  constant 
p  (u  +  v)  in  Table  XXVI  we  must  infer  that  the  valency  of  each 
of  these  ions  is  4.  These  phenomena  obviously  correspond  with 
those  which  would  be  exhibited  by  a  salt  which  forms  and  dis- 
sociates in  accordance  with  equations  of  the  type: 

H  Cl 

.COH.N.  .COH++ 


v 

XCOH.NX  NCOH++ 

/  \ 
H    Cl 

and 


COH.Nv  .COH++      . 

)^  +  H2S04  =  R/  +S04 

COH.NX  NCOH++ 


242  ELECTROCHEMISTRY 

which  are  in  satisfactory  accord  with  the  high  diamino-acid 
content  of  the  salmin  molecule  (4). 

Summing  up  these  results,  therefore,  we  see  that  in  ike  forma- 
tion of  the  salts  of  proteins  with  acids  diamino  radicals  are  primarily 
concerned,  while  in  the  formation  of  the  salts  of  protein  with  bases 
dicarboxylic  radicals  are  primarily  concerned.  The  correspondence 
between  the  diamino-acid  content  of  a  protein  and  its  binding 
capacity  for  acids,  to  which  Kossel  has  drawn  attention  (loc.  cit.), 
and  that  between  the  dicarboxylic  acid  content  and  the  com- 
bining capacity  for  bases  to  which  I  have  drawn  attention  in 
the  latter  part  of  the  first  chapter  is  therefore  seen  to  be  not 
accidental  but  an  expression  of  an  essential  feature  of  the  union 
between  proteins  and  inorganic  acids  and  bases. 
I  3.  The  Dependence  of  the  Electrical  Conductivity  of  Solutions 
of  Protein  Salts  upon  the  Proportion  of  Inorganic  Acid  or  Base 
which  the  Salt  Contains.  —  In  preceding  paragraphs  we  have 
discussed  the  effects  of  dilution  upon  the  conductivities  of  solu- 
tions of  protein  salts;  that  is,  of  altering  the  ratio  — j—  while 

keeping  the  composition  of  the  salt;  i.e.,  the  proportion  of  protein 
to  base  or  acid  constant.  We  have  found  that  these  effects 
admit  of  interpretation  in  the  light  of  the  well-known  laws  of 
electrolytic  dissociation  and  of  the  view  that  the  dissociation 
of  protein  salts  is  accomplished  by  the  splitting  of  —  COH.N  — 
bonds.  We  must  now  take  up  the  consideration  of  the  effects 
of  altering,  not  only  the  total  concentration  of  the  system,  but 
also  the  proportion  of  protein  to  base  or  acid. 

If  we  add  protein  to  dilute  solutions  of  an  acid  or  base  there 
usually  results  a  depression,  which  we  shall  designate  by  X,  of 
the  conductivity  of  the  solution.  I  find  that  when  1  per  cent  of 
casein  is  added  to  varying  concentrations  of  KOH  this  depression 
is  connected  with  the  concentration  of  the  KOH  solution  (=  61) 
to  which  the  protein  is  added  by  a  very  simple  relation.  This 
relation  is  of  the  form 

X  X  10-5  =  a&i  -  /36i2  -  7  (vi) 

in  which  a,  /?  and  7  are  constants  (14). 

Applying  this  formula  to  observations  in  which  1  per  cent  of 
casein  was  dissolved  in  varying  concentrations  of  KOH  *  and  the 

*  These  solutions  were  the  same  as  those  employed  in  the  gas-chain  measure- 
ments cited  in  the  previous  chapter  (1  per  cent  casein,  second  series),  and, 


PROPORTION  OF  INORGANIC  RADICAL 


243 


conductivities  of  the  solvent  and  of  the  solution  were  determined 
at  30  degrees,  and  computing  the  most  probable  values  of  the 
constants  a,  /3  and  7  from  all  of  the  observations  by  the  method 
of  least  squares,  we  obtain : 

X  X  105  =  26880  bi  -  475800  bj  -  28.98. 

In  the  accompanying  table  (Table  XXVIII)  the  experimental 
values  of  X  X  10~5  for  1  per  cent  solutions  of  casein  in  KOH  of 
various  concentrations  (=  &i)  and  those  calculated  from  the 
above  formula  are  compared.  In  the  first  column  are  given  the 
alkalinities  of  the  solutions  to  which  casein  was  added  (=  61); 
in  the  second  are  given  the  values  of  X  X  105  experimentally 
ascertained;  in  the  third  the  calculated  values  of  X  X  105;  in  the 
fourth  the  difference  (=  A)  between  the  experimental  and  the 
calculated  values  of  X  X  105;  and  in  the  fifth  the  possible  metrical 
error  (=  e)  in  the  experimental  determination  of  X  X  105. 

TABLE  XXVIII 


61 

XX105 

(experimental) 

XX  105 
(calculated) 

A 

€ 

0.03000 

348.8 

349.3 

+0.5 

±8.0 

0.02500 

343.0 

345.6 

+2.6 

±6.5 

0.02000 

322.1 

318.3 

-3.8 

±5.5 

0.01750 

299.7 

295.9 

-3.8 

±5.1 

0.01500 

268.2 

267.2 

-1.0 

±4.7 

0.01250 

230.1 

232.8 

+2.7 

±4.4 

0.01000 

186.8 

192.3 

+5.5 

±4.1 

0.00750 

141.6 

145.8 

+4.2 

±4.0 

0.00500 

92.1 

93.5 

+1-4 

±3.8 

0.00250 

43.9 

35.4 

-8.5 

±3.7 

SA  =  -0.2 

It  will  be  seen  that  the  deviations  of  the  calculated  from  the 
experimental  values  of  X  X  105  are  nearly  always  less  than  the 
possible  error,  due  to  instrumental  sources  alone,  in  the  experi- 
mental determination  of  X  X  105,  while  the  algebraic  sum  of  these 
deviations  is  negligible.  The  formula  therefore  represents,  in  a 
highly  satisfactory  manner,  the  relation  between  61  and  X  for 
1  per  cent  solutions  of  casein  in  KOH-solutions. 

therefore,  contained  varying  amounts  of  KC1  (Cf.  Appendix).  The  fact  that 
the  irregularity  in  KCl-content  does  not  disturb  the  regularity  of  the  relation 
between  X  and  61  is  further  proof  (Cf .  Chap.  VIII,  1),  that  KC1,  in  moderate 
concentrations,  does  not  appreciably  influence  the  conductivity  of  potassium 
caseinate  solutions. 


244 


ELECTROCHEMISTRY 


Investigating  the  similar  relations  which  obtain  in  solutions 
containing  different  percentages  of  casein  we  find  that  the  rela- 
tion between  X  and  61  for  all  of  the  concentrations  of  casein  in- 
vestigated can  be  represented  by  the  more  general  formula : 


X  X  105 


(vii) 


where  C  is  the  percentage  of  casein  and  a,  (3  and  y  are  constants, 
the  values  of  which  we  have  already  determined  for  1  per  cent 
casein.  For  a  0.5  per  cent  solution  of  casein  this  equation, 
therefore  becomes: 

X  X  105  =  26,880  61  -  951,600  6i2  -  14.49. 

In  the  following  table  the  experimental  and  calculated  values  of 
X  X  105  are  compared  —  the  symbols  have  the  same  meaning 
as  in  Table  XXVIII. 

TABLE  XXIX 


r 

XX10* 

XX10* 

01 

(experimental) 

(calculated) 

A 

6 

0.01000 

149.9 

159.2 

+  9.3 

±2.3 

0.00750 

123.5 

133.6 

+10.1 

±2.0 

0.00500 

88.2 

96.1 

+  7.9 

±1.8 

0.00250 

40.6 

37.6 

-  3.0 

±1.8 

0.00150 

23.3 

24.2 

+  0.9 

±1.8 

For  a  1.5  per  cent  solution  of  casein  equation  (vii)  becomes: 
X  X  105  =  26,880  61  -  317,200  bf  -  43.47. 

In  the  following  table  the  experimental  and  calculated  values 
of  X  X  105  are  compared: 

TABLE  XXX 


61 

XX106 
(experimental) 

XX  ID* 
(calculated) 

A 

6 

0.03000 

476.4 

477.5 

+    1.1 

±7.0 

0.02000 

366.5 

367.3 

+  0.8 

±5.1 

0.01500 

283.5 

288.4 

+  4.9 

±4.5 

0.01000 

188.5 

193.6 

+  5.1 

±4.1 

0.00750 

145.0 

140.4 

-  4.6 

±3.9 

0.00500 

95.4 

83.0 

-12.4 

±3.8 

PROPORTION  OF  INORGANIC  RADICAL 


245 


For  a  2.0  per  cent  solution  of  casein  equation  (vii)  becomes: 
X  X  105  =  26,880  61  -  237,900  6X2  -  57.96. 

In  the  following  table  the  experimental  and  calculated  values 
of  X  X  105  are  compared: 

TABLE  XXXI 


&1 

xxio* 

(experimental) 

xxio* 

(calculated) 

A 

€ 

0.05000 

661.6 

691.4 

+29.8 

±18.5 

0.03000 

539.3 

534.4 

-  4.9 

±11.5 

0.02000 

380.5 

384.5 

4-  4.0 

±  7.6 

0.01500 

286.2 

291.7 

4-  5.5 

±  7.1 

0.01000 

189.3 

187.1 

-  2.2 

±  6.7 

0.00500 

95.7 

70.5 

-25.2 

±  6.2 

For  a  3  per  cent  solution  of  casein  equation  (vii)  becomes: 
X  X  105  =  26,880  bi  -  158,600  6i2  -  86.94. 

In  the  following  table  the  experimental  and  calculated  values 
of  X  X  105  are  compared: 

TABLE  XXXII 


bi 

XX106 
(experimental) 

XXlQs 
(calculated) 

A 

.    € 

0.05000 

873.2 

860.7 

-12.5 

±16.4 

0.03000 

579.7 

576.8 

-  2.9 

±  9.0 

0.02500 

488.9 

486.0 

-  2.9 

±  8.4 

0.02000 

391.7 

387.3 

-  4.4 

±  7.6 

0.01500 

296.9 

280.6 

-16.3 

±  7.2 

0.01000 

206.6 

166.1 

-40.5 

±  6.8 

The  same  relation  holds  good  for  1.5  per  cent  solutions  of 
casein  in  Ca(OH)2  solutions  of  varying  concentration  (equivalent 
concentration  =  61)  (17). 

To  50  cc.  portions  of  a  3  per  cent  solution  of  casein  in  0.024 
N  Ca(OH)2  (neutral  to  phenolphthalein)  were  added  0,  2,  4,  6, 
or  12  cc.  of  0.048  N  Ca(OH)2  and  each  mixture  was  diluted  to 
100  cc.  Exactly  similar  solutions  were  made  up,  containing 
however,  no  casein.  The  conductivities  of  each  of  these  solu- 
tions were  then  determined  at  30  degrees.  Applying  equation 


246 


ELECTROCHEMISTRY 


(vii)  to  these  results  and  computing  the  constants  from  the  first, 
fourth  and  sixth  observations,*  we  obtain: 

X  X  105  =  30,830  61  -  488,000  &i2  -  53.4. 
In  the  following  table  the  experimental  and  calculated  values 
of  X  X  105  are  compared: 

TABLE  XXXIII 


61 

XX1Q5 
(experimental) 

XX  105 
(calculated) 

0.01200 

246.3 

246.3 

0.01296 

266.9 

264.2 

0.01392 

281.8 

281.1 

0.01488 

297.5 

297.5 

0.01584 

312.6 

312.5 

0.01776 

340.4 

340.4 

Formula  (vii)  also  applies  to  1  per  cent  solutions  of  ovomucoid 
in  varying  concentrations  of  KOH  and  HC1,  only  in  the  add 
solutions  we  must  replace  &i  by  ai}  the  acidity  of  the  solution 
to  which  the  ovomucoid  is  added.  For  1  per  cent  ovomucoid 
in  KOH  solutions,  computing  the  constants  by  the  method  of 
least  squares,  we  obtain: 

X  X  105  =  20,850  61  -  1,250,000  6i2  -  35.6. 

In  the  following  table  the  experimental  and  calculated  values 
of  X  X  105  are  compared: 

TABLE  XXXIV 


61 

XX  105 
(experimental) 

XXlQs 
(calculated) 

0.0005 

-26.8 

-25.5 

0.0010 

-16.0 

-16.0 

0.0020 

+  1.1 

+  1.1 

0.0030 

+15.7 

+15.7 

0.0050 

+38.2 

+38.2 

For  1  per  cent  ovomucoid  in  HC1  solutions,  computing  the 
constants  by  the  method  of  least  squares,  we  obtain: 
X  X  105  =  41,990  ai  -  852,700  ai2  -  41.4. 

*  These  results  do  not  lend  themselves  to  determination  of  the  constants  by 
the  method  of  least  squares,  since  the  normal  equations  are  nearly  identical, 
the  factors  differing  only  by  magnitudes  commensurate  with  the  experimental 
error. 


PROPORTION  OF  INORGANIC  RADICAL 


247 


In  the  following  table  the  experimental  and  calculated  values 
of  X  X  105  are  compared: 

TABLE  XXXV 


fll 

xxio5 

(experimental) 

XX1Q8 
(calculated) 

0.0005 

-  17.7 

-  20.6 

0.0010 

+    2.0 

-     0.3 

0.0020 

+  37.7 

+  39.2 

0.0030 

+  74.5 

+  76.9 

0.0040 

+111.1 

+113.0 

0.0050 

+146.2 

+147.2 

0.0075 

+228.9 

+225.5 

0.0100 

+291.8 

+293.2 

The  relation  does  not,  however,  hold  good  for  solutions  of 
casein  in  HC1.  Doubtless  it  would  hold  good  for  these  solutions 
also  were  it  possible  to  obtain  solutions  of  casein  in  dilute  HC1, 
without  at  the  same  time  introducing  the  chloride  of  the  base 
employed  to  dissolve  the  dry  casein.  But,  as  I  have  explained 
in  Chap.  V,  dry  casein  is  not  readily  dissolved  by  dilute  acids, 
and  in  order  to  secure  a  solution  of  casein  in  acid  it  is  necessary 
to  first  dissolve  the  casein  in  dilute  alkali,  and  then  add  acid  in 
excess  of  that  necessary  to  neutralize  the  base  and  sufficient 
to  redissolve  the  wet,  freshly  precipitated  casein.  Now  casein, 
dissolved  in  acids  is  very  markedly  influenced  in  its  solubility 
by  the  presence  of  salts;  the  departure  of  the  conductivities  of 
these  solutions,  therefore,  from  the  regularity  indicated  in  equa- 
tion (vii)  is  sufficiently  explicable. 

The  physico-chemical  significance  of  this  relation  is,  at  the 
present  stage  of  our  knowledge,  not  at  all  clear.  It  is  possible 
that  equation  (vii)  is  merely  an  interpolation-equation  and  that 
the  relation  is  of  purely  empirical  significance.*  It  is  chiefly  of 
use,  at  present,  as  we  shall  see,  in  throwing  light  upon  questions 

*  It  should  here  be  noted  that  any  relationship  of  this  type  must  involve 
the  mutual  dependence  of  the  following  factors: 

(i)  The  variation  of  m,  the  amount  of  acid  or  base  bound  by  the  protein 
with  variation  in  ai  or  61,  the  concentration  of  acid  or  alkali  in  which 
the  protein  is  dissolved. 

(ii)   The  variation  of  K  (the  dissociation-constant  of  the  salt)  and  possibly 
of  (u  +  t>)  with  the  quantity  of  acid  or  alkali  bound  by  the  protein, 
(iii)  The  effect  of  dilution  upon  the  conductivities  of  the  protein  salts. 


248  ELECTROCHEMISTRY 

involving  the  minimal  combining  capacity  of  proteins, '  such  as 
casein,  which  are  insoluble  in  the  free  condition. 

4.  The  Solubility  and  Minimal  Combining  Capacity  of 
Casein  and  of  Serum  Globulin  in  Solutions  of  Bases.  —  We  have 
seen  that  the  relation  between  61,  the  concentration  of  a  KOH- 
solution  in  which  casein  is  dissolved,  and  X,  the  depression  of 
its  conductivity  which  is  caused  by  the  casein,  are  connected 
by  the  relation 

X  X  10*  =  26,880  61  -  475'80(V  -  28.98  C; 

o 

putting  X  equal  to  zero  we  obtain 

61  =  0.00114  C  or  0.05536  C. 

Considering,  for  the  present,  only  the  smaller  value  of  61,  we 
see  that  when  X  =  0,  that  is,  when  the  change  in  the  conduc- 
tivity of  an  alkaline  solution  which  is  brought  about  by  dissolving 
a  given  percentage  of  casein  is  zero,  then  the  proportion  of  alkali 
to  casein  is  such  that  one  gram  of  casein  is  combined  with 
11.4  X  10~5  equivalents  of  alkali.  This  is  precisely  the  combining 
capacity  of  casein  at  "saturation"  of  the  base  with  casein,  that  is, 
when  the  base  has  dissolved  the  maximum  amount  of  casein  which 
it  will  dissolve. 

The  exact  coincidence  of  the  two  numerical  values,  especially 
when  we  consider  that  the  above  is  computed  by  least  squares 
from  a  number  of  determinations  which  are  apparently  not 
connected  with  estimates  of  the  solubility  of  casein,  is  surprising, 
and  leaves  no  room  for  doubt  that  the  magnitude  of  the  two 
quantities  is  determined  by  identical  factors.  This  result  is 
probably  to  be  interpreted  as  follows:  The  conductivity  (=  Zi) 
of  the  solution  of  base  to  which  casein  is  added  is  /u&i,  where 
fj,  is  the  equivalent  molecular  conductivity  of  the  base  and  61 
the  equivalent  concentration  of  the  base.  The  conductivity 
(=  x)  of  the  solution  containing  casein  is  vb  +  96.43  (u  +  0)  z, 
where  b  is  the  equivalent  concentration  of  the  unneutralized 
base,  u  +  v  is  the  sum  of  the  equivalent  mobilities  of  the  ions 
and  z  is  the  equivalent-  concentration  of  protein  ions.  Assuming 
that  at  the  concentrations  employed  the  casein  salt  is  tolerably 
completely  dissociated  z  =  pm,  where  m  is  the  equivalent  con- 
centration of  base  neutralized  by  the  protein  and  p  the  number 


SOLUBILITY  AND  COMBINING  CAPACITY  249 

of  equivalents  of  protein  salt  to  which  one  equivalent  of  base 
gives  rise.  Hence: 

X  =  xi  —  x  -  i*m  —  96.43  p  (u  +  v)  m. 

When  m  =  0,  therefore,  that  is,  when  the  quantity  of  base 
is  insufficient  to  combine  with  the  given  mass  of  casein,  X  must 
be  0.  Hence  the  value  of  61  when  X  =  0  is  a  true  measure  of  the 
minimum  combining  capacity  of  casein. 

For  1.5  per  cent  solutions  of  casein  in  calcium  hydrate  solutions 
we  found  that 

X  X  10-5  =  30,8306!  -  488,000  &i2  -  53.4; 
putting  X  =  0  and  estimating  61  we  find  that 

61  =  0.001782; 

dividing  this  by  1.5,  the  percentage  of  casein,  we  find  that  X  =  0 
when  the  equivalence  between  the  casein  and  the  calcium  hydrate 

18  1  gram  =  11.9  X  10~5  equivalents; 

a  value  so  near  that  (11.4  X  10~5)  obtained  for  the  alkalies  that 
they  may  be  regarded  as  being,  within  the  experimental  error, 
identical. 

In  the  previous  (German)  edition  of  this  work  I  stated  that 
we  were  justified  in  the  light  of  these  results  in  tentatively 
assuming,  pending  more  direct  evidence,  that  the  various  bases 
dissolve  casein  in  equivalent-molecular  proportions.  Since  then 
Van  Slyke  and  Bosworth  have  shown  (25),  by  direct  analytical 
determinations,  employing  dialysis  to  remove  the  chloride  formed 
on  neutralizing  the  excess  of  base  with  hydrochloric  acid,  that 
the  minimal  proportion  of  a  diacid  base  which  will  dissolve  casein 
is  about  23  X  10~5  equivalents  but  that  an  insoluble  compound 
is  formed  containing  11.3  equivalents  of  the  base.  In  other 
words  the  minimal  equivalent  combining  capacity  of  casein  for 
bases  is  the  same  for  diacid  as  monacid  bases. 

We  have  seen  (1)  that  each  —  COH.N—  bond  of  casein  which 
is  opened  up  by  the  entrance  of  a  KOH  molecule  reacts  with 
the  base  in  accordance  with  the  equation : 

H 

-COH.N-  +  KOH  =  COK  +  ^N- 

I 
OH 


250  ELECTROCHEMISTRY 

while  in  reacting  with  diacid  bases  two  —  COH.N—  groups  are 
involved,  part  of  the  valencies  of  the  ions  which  are  formed  suffer- 
ing internal  neutralization  thus: 

-CO++  H 

2  -COH.N-  +  Ca(OH)2  =  /Ca  +  ^N- 

/  I 

-CO.N-  OH 

/  \ 
H    OH 

If  this  view  be  correct,  then  one  molecule  of  calcium  hydrate 
must  obviously  bind  twice  the  weight  of  casein  that  is  bound 
by  one  molecule  of  KOH.  Hence  di-  and  monacid  bases  unite 
with  casein  in  equivalent-molecular  proportions,  but  at  "satu- 
ration" of  the  base  with  casein,  when  the  mass  of  the  protein 
ions  is  at  its  maximum,  the  cations  formed  as  a  result  of  the 
union  with  diacid  bases,  being  much  more  bulky  than  those 
formed  by  the  union  with  monacid  bases,  fail  to  pass  into  solu- 
tion. Hardy  (2)  has  found  that  bases  dissolve  serum-globulin, 
not  in  equivalent  molecular  but  in  molecular  proportions.  It  is 
probable  that  in  this  case  also  an  insoluble  salt  is  formed  with 
the  minimal  proportion  of  diacid  base. 


LITERATURE  CITED 

(1)  Bredig,  G.,  Zeit.  f.  physik.  Chem.  13  (1894),  p.  191. 

(2)  Hardy,  W.  B.,  Journ  of  Physiol.  33  (1905),  p.  251. 

(3)  Kossel,  A.,  Zeit.  f.  physiol.  Chem.  25  (1898),  p.  165. 

(4)  Kossel,  A.,  and  Dakin,  H.  D.,  Zeit.  f.  physiol.  Chem.  41  (1904),  p.  412. 

(5)  Osborne,  W.  A.,  Journ.  of  Physiol.  27  (1901),  p.  389. 

(6)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  2  (1907),  p.  337. 

(7)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  11  (1907),  p.  437. 

(8)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  11  (1907),  p.  542. 

(9)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  5  (1908),  p.  147. 

(10)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  12  (1908),  p.  473. 

(11)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  13  (1909),  p.  469. 

(12)  Robertson,  T.  Brailsford,  "The  Proteins,"  Univ.  of  California,  Publ. 

Physiol.  3  (1909),  p.  115. 

(13)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  14  (1910),  p.  377. 

(14)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  14  (1910),  p.  528. 

(15)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  14  (1910),  p.  601. 

(16)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  14  (1910),  p.  709. 

(17)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  15  (1911),  p.  166. 


LITERATURE  CITED  251 

(18)  Robertson,  T.  Brailsford,  "Die  physikalische  Chemie  der  Proteine," 

Dresden  (1912). 

(19)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  16  (1912),  p.  382. 

(20)  Robertson,  T.  Brailsford,  and  Burnett,  T.  C.,  Journ.  Biol.  Chem.  6 

(1910),  p/105. 

(21)  Sackur,  O.,  Zeit.  f.  physik.  Chem.  41  (1902),  p.  672. 

(22)  Sutherland,  W.,  Proc.  Roy.  Soc.  London,  79  B  (1907),  p.  130. 

(23)  Sutherland,  W.,  Phil.  Mag.  6  Ser.  14  (1907),  p.  1.. 

(24)  Taylor,  A.  E.,  Univ.  of  California  Publ.  Pathol.  1  (1904),  p.  7. 

(25)  Van  Slyke,  L.  L.,  and  Bosworth,  A.  W.,  Journ.  Biol.  Chem.  1  (1913), 

p.  211. 


CHAPTER  XI 
THE  ELECTROCHEMISTRY  OF  COAGULATION 

1.  The  Coagulation  of  the  Casemates  by  Alcohol.  —  If  one- 
half  a  cubic  centimeter  of  a  0.0125  N  solution  of  KOH,  neutralized 
either  to  phenolphthalein  or  to  litmus  by  the  addition  of  casein, 
be  added  to  10  cc.,  i.e.,  to  20  volumes  of  99.8  per  cent  alcohol, 
no  coagulation  of  the  protein  occurs,  although  the  solution  which 
is  thus  obtained  is  appreciably  more  opalescent  than  a  solution 
of  equal  concentration  in  water  containing  no  alcohol.  Even 
if,  instead  of  employing  a  solution  of  potassium  caseinate  in  water, 
we  employ  a  0.0125  N  solution  of  KOH  in  75  per  cent  alcohol, 
containing  1.6  or  2.5  per  cent  of  casein,  adding  J  cc.  of  this  to 
10  volumes  of  99.8  per  cent  alcohol,  still  no  coagulation  of  the 
caseinate  occurs,  although  it  is  now  dissolved  or  forms  a  stable 
suspension  in  a  98.6  per  cent  solution  of  alcohol.  The  caseinate 
can,  however,  be  readily  coagulated  by  adding  to  this  mixture 
an  equal  volume  of  ether  and  allowing  it  to  stand  for  a  few  hours. 

Very  different  is  the  behavior  of  the  caseinates  of  the  alkaline 
earths.  If  to  10  cc.  each  of  60,  70,  75  per  cent,  etc.,  solutions  of 
alcohol  we  add  J  cc.  of  a  0.012  N  solution  of  Ca(OH)2,  neutralized 
to  phenolphthalein  by  casein,  distinct  coagulation  of  the  casein- 
ates occurs,  on  shaking,  when  the  final  concentration  of  alcohol 
in  the  mixture  is  about  55  per  cent.  The  same  is  true  for  an 
equally  concentrated  solution  of  barium  hydroxide  neutralized 
to  phenolphthalein  by  casein.  For  strontium  caseinate  the 
limiting  concentration  of  alcohol  at  which  coagulation  occurs  is 
much  higher,  about  70  per  cent.  In  all  cases  coagulation  is  much 
accelerated  by  energetic  shaking  of  the  mixture. 

It  is  of  interest  to  compare  the  marked  difference  between  the 
behavior  of  the  caseinates  of  the  alkalies  and  of  the  alkaline  earths 
upon  the  addition  of  alcohol  to  their  aqueous  solutions,  with  the 
numerous  other  differences  which  subsist  between  the  caseinates 
of  the  alkalies  and  those  of  the  alkaline  earths. 

Thus  the  aqueous  solutions  of  the  "neutral"  or  "basic"  casein- 

252 


COAGULATION  BY  ALCOHOL  253 

ates  of  the  alkalies  and  ammonium  are  clear,  do  not,  except 
lithium  caseinate,  show  any  increase  in  turbidity  on  warming, 
and  are  not  precipitated  by  the  addition  of  finely  divided  in- 
soluble substances  or  by  passing  through  a  clay  filter;  while 
the  aqueous  solutions  of  the  caseinates  of  the  alkaline  earths 
are  opalescent,  show  marked  increase  in  turbidity  on  heating 
their  solutions  to  35-45°  C.  which  disappears  on  cooling,  and  are 
precipitated  from  their  solutions  by  the  addition  of  finely  divided 
insoluble  substances  or  by  passage  through  a  clay  filter  (4). 

The  " neutral"  or  " basic"  casemates  of  the  alkalies,  upon 
solution  in  water,  depress  its  freezing  point  and  the  amount 
of  the  depression  is  that  of  a  solution  of  the  same  molecular 
concentration  as  the  alkaline  solution  which  is  employed  as 
solvent.  The  depression  produced  by  calcium  caseinate  is  much 
less,  being  less  than  half  as  great  (10). 

Equally  concentrated  solutions  of  the  hydroxides  of  the  alka- 
lies and  ammonium  dissolve  casein  at  approximately  the  same 
rate.  Solutions  of  the  hydroxides  of  the  alkaline  earths  dissolve 
casein  much  more  slowly  (8).  The  rate  of  the  solution  of  casein 
by  solutions  of  the  hydroxides  of  the  alkalies  is  accelerated  by 
raising  the  temperature  above  36  degrees;  the  rate  of  the  solu- 
tion of  casein  by  solutions  of  calcium  hydroxide  is  materially 
diminished  by  a  similar  rise  in  temperature  (6). 

The  caseinates  of  the  alkaline  earths  are  precipitated  from  their 
solutions  by  the  addition  of  small  concentrations  of  the  chloride 
of  the  corresponding  alkaline  earth  (7);  the  caseinates  of  the 
alkalies  are  not  precipitated  by  the  addition  of  similar  quan- 
tities of  the  chlorides  of  the  alkalies. 

I  have  alluded  to  the  fact  that  the  limiting  concentration  of 
alcohol  at  which  strontium  caseinate  is  coagulated  is  consider- 
ably higher  than  that  at  which  calcium  and  barium  caseinates 
are  coagulated.  This  is  curious  because  we  should  expect,  as 
a  rule,  to  find  the  salts  of  strontium  possessed  of  properties  in- 
termediate between  those  of  calcium  and  barium.  It  is  therefore 
the  more  interesting  to  observe  that  this  is  not  the  only  respect 
in  which  the  caseinates  of  strontium  exhibit  behavior  differing 
from  that  of  the  caseinates  of  calcium  and  barium  and  approach- 
ing the  behavior  of  the  caseinates  of  the  alkalies;  for  casein 
dissolves  much  more  rapidly  in  solutions  of  strontium  hydroxide 
than  in  solutions  of  other  alkaline  earth  hydroxides  (8);  the 


254  ELECTROCHEMISTRY 

solutions  of  strontium  caseinate  are  less  opalescent  than  equally 
concentrated  solutions  of  calcium  or  barium  caseinate,  and  the 
dissociation-constant  of  "basic"  strontium  caseinate  is  much 
larger  than  those  of  barium  or  calcium  caseinate,  and  is  inter- 
mediate in  magnitude  between  these  and  the  dissociation  con- 
stants of  the  " basic"  caseinates  of  sodium  and  ammonium. 
This,  however,  is  not  a  general  phenomenon  where  protein  salts 
of  the  alkaline  earths  are  concerned,  because  the  dissociation 
constant  for  the  strontium  salt  of  "insoluble"  serum  globulin 
is  not  very  appreciably  different  from  those  of  the  calcium  and 
barium  salts  (Cf.  previous  chapter). 

The  caseinates  of  the  alkalies  and  alkaline  earths  dissolved  in 
various  concentrations  of  alcohol  afford,  therefore,  very  favor- 
able material  for  the  investigation  of  the  intimate  nature  of  the 
process  of  coagulation,  since  different  caseinates,  in  the  presence 
of  the  same  quantity  of  alcohol,  may  be,  to  all  appearance  com- 
pletely soluble,  or  completely  coagulated,  and  as  we  shall  see,  a 
comparison  of  the  behavior  of  these  different  caseinates  towards 
the  presence  of  varying  percentages  of  alcohol  in  their  solutions 
leads  to  a  very  considerable  insight  into  the  chemical  mechanics  of 
protein-coagulation  by  alcohol. 

2.  The  Applicability  of  the  Ostwald  Dilution-Law  to  Solutions 
of  the  Casemates  in  Alcohol-Water  Mixtures.  —  In  the  preceding 
chapters  it  has  been  shown  that  the  Ostwald  dilution-law  for  a 
binary  electrolyte  written  in  the  form 

1.037  X  10-2     .   1.075  X  10-4  2  ,., 

m  =  — / — i — \~x  H — TTI — i — \rx  (0 

p  (u  +  v)  PK  (u  +  v)2 

is  applicable  to  many  aqueous  solutions  of  the  salts  which  pro- 
teins form  with  inorganic  acids  and  bases,  m  being  the  equivalent 
molecular  concentration  of  the  acid  or  base  which  is  combined 
with  the  protein,  p  the  number  of  equivalents  of  protein  salt 
to  which  one  equivalent  of  neutralized  acid  or  base  gives  rise, 
x  the  conductivity  of  the  solution  in  reciprocal  ohms  per  cubic 
centimeter,  K  the  dissociation  constant  of  the  salt  and  u  +  v 
the  sum  of  the  equivalent  specific  migration-velocities  of  the 
ions  at  infinite  dilution.  The  same  law  applies  to  solutions  of 
potassium  caseinate  (80  X  10~5  equivalents  KOH  per  gram)  in 
alcohol-water  mixtures  which  contain  up  to  and  less  than  60 
per  cent  alcohol,  as  the  following  experimental  results  show  (9). 


DILUTION-LAW  255 

In  five  hundred  cubic  centimeters  of  N/ 10  KOH  were  dissolved 
62.5  grams  of  casein,  thus  forming  a  solution  of  potassium  casein- 
ate,  neutral  to  phenolphthalein.  To  50  cc.  portions  of  this 
solution  were  added,  respectively,  0,  20,  40,  etc.,  cubic  centi- 
meters of  alcohol,  and  the  mixtures  were  diluted  to  200  cc.  with 
water.  Each  of  these  solutions  was  then  diluted,  by  the  addition 
of  alcohol  of  the  same  concentration,  to  the  desired  concentra- 
tions of  casein.  All  alcohol  percentages  are  percentages  of  99.8 
per  cent  alcohol  by  volume.  The  solutions  in  "75  per  cent 
alcohol"  were  made  up  by  adding  99.8  per  cent  alcohol  to  50  cc. 
of  the  original  solution  of  caseinate  until  the  volume  was  200  cc., 
and  diluting  with  a  75  per  cent  by  volume  solution  of  99.8  per 
cent  alcohol.  The  conductivity  determinations  were  made  at 
30°  C.  The  conductivity  of  the  distilled  water  (=  4  X  lO"6) 
has  been  subtracted  from  each  of  the  tabulated  conductivities. 
The  results  are  given  in  Table  I  on  the  following  page. 

Applying  the  above  equation  to  these  results  and  computing 

1.037  X  10-2        ,   1.075  X  10-5   . 

the  constants   — -, — : — r—  and  —^-, — : — ^   for  each  concen- 
p  (u  +  v)  pK  (u  +  v)z 

tration  of  alcohol  from  all  of  the  results  by  the  method  of  least 
squares,  we  obtain: 

For  the  solutions  containing  0  per  cent  of  alcohol: 

m  =  12 .41  x  +  1666  x2.  (ii) 

For  the  solutions  containing  10  per  cent  of  alcohol: 

m  =  15.97  x  +  3325  x2.  (iii) 

For  the  solutions  containing  20  per  cent  of  alcohol: 

m  =  21.14z  +  5880z2.  (iv) 

For  the  solutions  containing  30  per  cent  of  alcohol: 

m  =  27.23  x  +  10,858  x2.  (v) 

For  the  solutions  containing  40  per  cent  of  alcohol: 

m  =  34.63  x  +  18,931  x\  (vi) 

For  the  solutions  containing  50  per  cent  of  alcohol: 

m  =  42.91  x  +  35,937z2.  (vii) 

For  the  solutions  containing  60  per  cent  of  alcohol: 

m  =  53.49  x  +  78,335  x2.  (viii) 

For  the  solutions  containing  75  per  cent  of  alcohol : 

m  =  29.95  x  +  1,219,800  x9.  (ix) 


256 


ELECTROCHEMISTRY 


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CONCENTRATION  OF  ALCOHOL  257 

Inserting  the  observed  values  of  x  in  equations  (ii)  to  (ix)  ,  we  can 
compute  the  "theoretical"  values  of  m,  that  is,  the  equivalent 
concentration  of  KOH  neutralized  by  the  casein  which  should, 
provided  the  dilution-law  holds  good,  correspond  to  the  observed 
conductivities.  In  Table  II,  on  the  following  page,  the  observed 
and  calculated  values  of  m  are  compared. 

It  is  evident  that  the  correspondence  between  the  experimental 
results  and  those  which  are  indicated  by  the  Ostwald  dilution- 
law  for  a  binary  electrolyte  is  all  that  could  be  desired  for  the 
solutions  containing  from  0  to  60  per  cent  of  alcohol.  For  the 
solutions  containing  75  per  cent  of  alcohol  the  divergences  be- 
tween theory  and  experiment  are,  however,  considerably  greater 
than  could  be  accounted  for  by  experimental  error  and  probably 
indicate  that  the  law  does  not  hold  good  for  these  solutions.  The 
sudden  alteration  in  the  relative  values  of  the  constants  which 
occurs  when  the  percentage  of  alcohol  attains  this  magnitude 
indicates,  whether  the  Ostwald  law  be  considered  as  holding 
good  or  not,  a  profound  change  in  the  molecular  and  ionic  con- 
dition of  the  protein. 

It  would  appear,  however,  that  we  are  justified  in  concluding 
that  potassium  caseinate  dissociates  in  alcohol-water  mixtures 
containing  from  0  to  60  per  cent  of  alcohol  in  the  same  manner 
as  it  does  in  aqueous  solutions. 

3.  The  Influence  of  the  Concentration  of  Alcohol  in  the  Solvent 
upon  the  Conductivities  of  Solutions  of  Potassium  Caseinate.  — 
I  find  (9)  that  the  conductivity  (=  xv)  of  a  solution  containing 
any  given  concentration  of  casein  combined  with  KOH  in  the 
proportion  requisite  to  secure  neutrality  to  phenolphthalein,  and 
dissolved  in  an  alcohol-water  mixture  containing  y  per  cent  of 
alcohol  by  volume  is  connected  with  the  conductivity  (=z0) 
of  a  similar  solution  in  water  by  the  formula 

XQ 


where  A  is  a  constant  which  varies  but  slightly,  if  at  all,  with  the 
concentration  of  the  caseinate.  This  constant  is  determined  in 
the  following  manner:  If  the  logarithms  to  base  10  of  x  corre- 
sponding to  y  =  0,  10,  20,  30,  40,  50,  and  60  per  cent  be  tabulated 
in  that  order  and  each  successive  value  of  logo;  be  subtracted 
from  the  one  lying  immediately  above  it,  the  differences  thus 


258 


ELECTROCHEMISTRY 


1     ++ 


si 


»oco  I-H  co 

(MCOCOT-H 


xi 

8*1 


CD  C^  OO  T-H 
•  co  CO  C<l  T-I 


w 


"5  CO  T-H  CO 
<M  COCOT-i 


TH  0$   TH   C<1   TH 

\  \  \++ 

38S8TH 


si 

xl 


rH  r-H  r—  !  CO  r—  1 

4-  1   1  ++ 


- 


3 

3 


C<J 


CO  rH  CO 

COCO  TH 


>OCO  i-H  CO 
(M  COCO  T-I 


1    ++ 


+ 


;^s^2 


O  TH  1—  ICO  TH 

1  1  ++ 


>o  co 

OQ  CO 


si 


OC^OCOrH 

I    ++ 

SS5S^5 


i—  I  CO 


^OCO  TH  CO 
1NCOCOTH 


CONCENTRATION  OF  ALCOHOL 


259 


computed  are  observed  to  be  appreciably  constant  and  equal 
to  ten  times  the  log  to  base  10  of  A  in  the  above  equation.  The 
average  value  of  A  for  all  of  the  determinations  enumerated  in 
Table  I  (omitting,  in  the  computation,  the  solutions  containing 
75  per  cent  alcohol)  is  1.0265.  For  the  various  concentrations 
of  potassium  caseinate  the  values  of  A  are  as  follow: 

TABLE  III 


Concentration  of  KOH 

L  neutralized  by  casein 

A 

0.0250 
0.0125 
0.0063 
0.0031 
0.0016 

1.0279 
1.0270 
1.0265 
1.0252 
1.0257 

Average  1.0265 

In  the  accompanying  table  (Table  IV)  the  experimental  values 
of  x  and  those  computed  from  the  formula 


XQ        A" 

are  compared.  Two  "calculated"  values  of  xy  are  given,  the 
first  calculated  value  of  xy  being  computed  from  the  average 
value  of  A  for  the  given  concentration  of  caseinate,  the  second 
being  computed  from  the  average  value  of  A  for  all  the  deter- 
minations enumerated  in  Table  I,  with  the  omission,  of  course, 
of  those  for  the  solvent  containing  75  per  cent  of  alcohol. 

The  same  relation  also  holds  good  for  solutions  of  "  neutral" 
potassium  caseinate,  that  is,  for  solutions  which  contain  50  X  10~5 
equivalents  of  base  per  gram  and  are  neutral  to  litmus  instead 
of;  as  in  the  experiments  cited  above,  80  X  10~5  equivalents  of 
base  per  gram  (neutral  to  phenolphthalein).  Two  and  a  half 
per  cent  solutions  of  casein  in  0.0125  N  KOH  were  prepared, 
containing  0,  10,  20,  50,  and  75  per  cent  of  alcohol  respectively 
The  relation  as  we  shall  see,  does  not  hold  good  for  the  solution 
containing  75  per  cent  alcohol  and  so,  in  computing  the  value 
of  A  for  these  solutions,  we  must  omit  the  determination  for  the 
solution  containing  75  per  cent  alcohol.  The  average  difference 
in  logio  xy  for  10  per  cent  increase  in  y  is  computed  by  adding 


260 


ELECTROCHEMISTRY 


II 

9.J3 


&  . 


ecooocoiococcc^i 


E  a 
O'S 

MI 


s 


P 

B* 


X| 


coooecoooo^o 


"50i»OrH 


iO  O  >O  C 


x 


xi 


x 


CONCENTRATION  OF  ALCOHOL 


261 


together  the  three  observed  differences  (between  xv  corresponding 
to  y  =  0  and  10  per  cent;  10  and  20  per  cent;  20  and  50  per 
cent)  and  dividing  their  sum  by  5.  In  this  way  A  was  ascer- 
tained to  possess,  for  these  solutions  (at  30  degrees)  the  average 
value  1.0248.  In  the  following  table  the  actual  and  computed 
values  of  xv  are  compared : 

TABLE  V.    CONCENTRATION  OF  KOH  NEUTRALIZED  BY 
CASEIN  0.0125  N 


V 

ZyXlO8 

(observed) 

z-XlO* 
(calculated) 

0 

87 

87 

10 

66 

68 

20 

51 

53 

50 

25 

25 

75 

8 

14 

It  is  evident  that  the  law  holds  good  for  solutions  containing 
from  0  to  60  per  cent  of  alcohol  inclusive,  but  that  it  does  not 
hold  good  for  solutions  containing  75  per  cent  alcohol,  since  for 
these  solutions  the  calculated  and  observed  values  of  xv  are 
widely  divergent.  Moreover  the  law  does  not  hold  good  for 
solutions  of  the  caseinates  of  the  alkaline  earths  in  alcohol-water 
mixtures;  this  is  evidenced  by  the  lack  of  constancy  in  the  first 
differences  of  logic  z  corresponding  to  equal  increments  of  y.  In 
the  accompanying  table  are  given  the  values  of  logio  #  correspond- 
ing to  various  values  of  y  for  solutions  of  calcium  caseinate  at 
30  degrees,  containing  1.5  per  cent  of  casein  combined  with 
80  X  10~6  equivalents  of  base  per  gram. 

TABLE  VI 


y  =  per  cent  of  alco- 
hol by  volume 

log,0  *  X  10« 

Differences 

0 
10 
20 
30 
40 
50 

1.24797 
0.87506 
0.56820 
0.34242 
0.20412 
0.07918 

0.37291 
0.30686 
0.22578 
0.13830 
0.12494 

In  the  following  table  are  given  the  corresponding  data  for  solu- 
tions of  strontium  caseinate  at  30  degrees,  containing  1.5  per 


262 


ELECTROCHEMISTRY 


cent  of  casein  combined  with  80  X  10~5  equivalents  of  base  per 
gram  i 

TABLE  VII 


y  =  per  cent  of  alco- 
hol by  volume 

logio  x  X  108 

Differences 

0 
10 
20 
30 
40 
50 

1.39967 
1.09342 
0.79934 
0.62325 
0.50515 
0.36173 

0.30625 
0.29408 
0.17609 
0.11810 
0.14342 

It  is  perhaps  not  without  significance  that  in  the  above  table 
the  first  two  differences  are  much  more  alike  than  the  succeeding 
differences.  In  solutions  containing  0  to  20  per  cent  of  alcohol, 
strontium  caseinate  is  further  from  coagulation  than  correspond- 
ing solutions  of  calcium  caseinate  and,  as  we  have  seen,  A,  in 

>y» 

the  equation  xy  =  -j-,  approaches  constancy.    Having  regard  to 

the  observed  failure  of  the  solutions  of  potassium  caseinate  in 
75  per  cent  alcohol  to  conform  to  the  law,  and  to  the  fact  that 
at  an  alcohol-concentration  lying  between  60  and  75  per  cent, 
solutions  of  potassium  caseinate  undergo  a  great  and  relatively 
sudden  increase  in  opacity,  we  may,  I  think,  conclude  that  the 
coagulation  of  a  caseinate  by  alcohol  is  heralded  by  a  failure  of  the 

solution  to  conform  to  the  law  xy  =  ~^,  connecting  the  percentage  of 
alcohol  in  the  solution  with  its  conductivity. 

4.  The  Interpretation  of  the  Law  xv  =  ^ .  —  As  we  have 

observed  (Table  III)  the  value  of  A  is  not  only  constant  for  all 
dilutions  of  potassium  caseinate,  but  also  is  very  nearly  constant 
for  all  of  the  proportions  of  alcohol  to  water  employed  as  solvents. 
Provided  A  were  rigorously  constant  we  would  obviously  obtain 
the  relation: 


Sale.  H2O 


constant 


for  any  given  proportion  of  alcohol  to  water.  This  is  the  rela- 
tion, suggested  by  Cohen  (2),  connecting  the  conductivities  of 
inorganic  salts  in  mixtures  of  alcohol  and  water.  Roth  (12), 
however,  found  that  this  rule  does  not  strictly  hold  good  for 


INTERPRETATION  263 

solutions  of  KC1  in  mixtures  of  alcohol  and  water,  but  that  the 


quotient   —         decreases  somewhat  with  increasing  dilution. 

Zalc.H80 

In  the  accompanying  table  (page  264)  the  values  of  —  2^—  are 

Zalc.  H20 

given  for  solutions  of  potassium  caseinate  (containing  80  X  10~5 
equivalents  of  base  per  gram,  Cf.  Table  I)  containing  each  of  the 
proportions  of  alcohol  to  H2O  employed. 

It  will  be  observed  that  the  values  of  —  -^-  are  appreciably 

Zalc.  H,0 

constant,  for  each  proportion  of  alcohol  to  water,  for  the  solu- 
tions containing  0  to  60  per  cent  of  alcohol,  but  that  in  the  solu- 
tions containing  75  per  cent  of  alcohol  the  value  of  this  ratio 
does  not  even  approximate  to  constancy  but  decreases  rapidly. 
There  is,  it  is  true,  a  slight  but  regular  diminution  in  the  value 
of  this  ratio  even  in  the  solutions  containing  0  to  60  per  cent  of 
alcohol,  in  passing  from  an  equivalent  concentration  of  neutral- 
ized KOH  of  0.0250  to  0.0016,  but,  except  in  the  solutions  con- 
taining 75  per  cent  of  alcohol,  the  diminution  is  not  more  than 
a  few  per  cent. 

We  have  seen  (equations  (ii)  to  (ix))  that  the  Ostwald  dilution- 
law,  in  the  form  expressed  in  equation  (i)  holds  good,  at  any 
rate  for  solutions  of  potassium  caseinate  in  alcohol-water  mix- 
tures containing  0  to  60  per  cent  of  alcohol. 

From  the  symmetry  of  the  equation: 

1.037  X  lO-2      .   1.075  X  10~4 

m  —  -  7  -  i  -  \  —  x    i  --  TT~i  -  i  -  \T~  x  t 

p(u-\-v)  PK  (u  +  t?)2 

it  would  follow,  were  A  s'trictly  constant  for  all  dilutions,  that 
the  proportions  of  alcohol  to  water  employed  as  solvent  for  the 
caseinate  affects  only  u  +  v,  i.e.,  the  migration-velocities  of  the 
ions,  and  not  the  dissociation-constant  (=  K)  or  the  number  of 
equivalents  of  caseinate  resulting  from  the  neutralization  of  one 
molecule  of  KOH  (=  p).*  Since  the  alteration  in  A  with  dilu- 
tion of  the  potassium  caseinate  is  so  small  for  solvents  containing 

*  For,  since  x  appears  in  one  term  of  the  equation  as  the  first  power,  and 


in  the  second  as  the  square,  if  the  ratio  of    —  »_    is  constant  any  other 

xalc.  H,O 

factor  in  the  equation  which  is  affected  by  the  presence  of  alcohol  must  also 
appear  in  the  one  term  of  the  equation  as  the  first  power  and  in  the  other  as 
the  square. 


264 


ELECTROCHEMISTRY 


ncen 
raliz 
in 


a 
^W 

I 


bo'do'd 


ft 

•gM 


I 

J* 


o  icco  i— i  co 
ddodd 


CO  >-H  i—  i  O  OS 
CO  CO  CO  CO  C3 


OjOCOj-HCO 
iO  (N  CO  CO  i— i 
CO  T-H  O  O  O 

ddddd 


a 
SW>> 


111 


ill 

•32S 


O  Is-  Oi  00  O 

I>T^  T-H  OiOi 

CQ  OS  GO  «D  CO 


o  *o  co  I-H  co 

00000 


»O  -^  Tt<  rt<  rj< 


oo'ooo 


E|l 

IS  s 


co  co  co  co  co 


SSS88 

ddddd 


ddddd 


INTERPRETATION 


265 


0  to  60  per  cent  of  alcohol,  we  may  conclude  that  in  these  solu- 
tions it  is  for  the  greater  part  the  migration-velocity  of  the 
caseinate  ions  which  is  affected  by  the  percentage  of  alcohol 
in  the  solvent.  From  the  values  of  the  constants  in  equations 
(iii)  to  (ix)  it  is  apparent  that  the  migration-velocities  of  the 
casein-ions  are  progressively  diminished  by  increasing  alcohol- 
concentration  from  0  to  60  per  cent.  From  the  facts  that  the 

law  xv  =  ~  no  longer  holds  good  for  the  solutions  containing 

Ay 

75  per  cent  alcohol  and  the  ratio  — ^*°_  is  not  constant  we  may 

Zalc.H,O 

conclude  that  in  these  solutions  the  degree  of  dissociation  of  the 
caseinate  or  the  number  of  equivalents  of  casein  produced  by 
one  equivalent  of  KOH  or  both  undergo  a  profound  modification. 
The  values  of  the  constants  in  equation  (x)  indicate  an  increase 
in  the  magnitude  of  u  +  v  and  a  very  great  decrease  in  the  mag- 
nitude of  the  dissociation-constant.  In  the  following  table  the 

Tr 

values  of  p  (u  +  v)  and  of  —  corresponding  to  the  equations  (iii) 

$  P 

to  (x)  are  enumerated;  in  the  fourth  column  is  given  the  per- 
centage dissociation  of  the  caseinate  in  the  solutions  containing 
0.025  N  KOH  bound  by  casein  (at  30  degrees). 

TABLE  IX 

Potassium  Caseinate.    80X10"5  Equivalents  of  KOH  per  gram 


Per  cent  of  alcohol 
by  volume 

"+< 

K 

p 

Per  cent  of  caseinate 
dissociated  in  solutions 
containing  0.025  equiv- 
alent of  potassium 

0 
10 
20 
30 
40 
50 
60 
75 

83.6X10-6 
64.9XHT6, 
49.1X10-* 
38.0X10"6 
29.9X10-6 
24.2XHT6 
19.4X10-6 
34.6X10-8 

0.0923 
0.0767 
0.0760 
0.0682 
0.0633 
0.0513 
0.0365 
0.0007 

82 
80 
79 
78 
77 
74 
68 
17 

The  degree  of  dissociation  of  the  caseinate  in  the  solutions  con- 
taining 60  per  cent  and  less  than  60  per  cent  of  alcohol  is  evidently 
but  little  affected  by  the  percentage  of  alcohol  in  the  solvent,  the  major 
part  of  the  effect  of  the  alcohol  upon  the  conductivity  being  attri- 
butable to  the  decreased  mobility  of  the  caseinate  ions. 


266  ELECTROCHEMISTRY 

5.  The  Viscosities  of  Solutions  of  Potassium  Caseinate  in 
Alcohol-Water  Mixtures.  —  The  striking  resemblance  between 

the  formula  —  =  -j-  and  the  Arrhenius-Euler  formula,  —  =  an, 

XQ         A.  y  TJQ 

for  the  dependence  of  the  viscosity  of  a  solution  upon  its  con- 
centration (1),  where  t\n  is  the  viscosity  of  the  solution,  770  that  of 
the  solvent,  n  the  concentration  of  the  solution  and  a  is  constant, 
forcibly  suggests  that  the  decrease  in  the  conductivity  of  potas- 
sium caseinate  solutions  due  to  the  addition  of  alcohol  between 
alcohol-percentages  of  0  to  60  may  be  attributable  for  the  greater 
part  to  the  hampering  of  the  protein  ions  by  the  increased  in- 
ternal friction  of  the  solvent.* 

Accordingly  the  following  determinations  were  made: 

31.25  grams  of  casein  were  dissolved  in  250  cc.  of  AT/10  KOH. 
To  25  cc.  portions  of  this  were  added  0,  10,  20,  etc.  cc.  of  alcohol, 
the  volume  of  each  mixture  being  made  up  to  100  cc.,  the  solution 
containing  75  per  cent  alcohol  being  made  up  by  adding  99.8  per 
cent  alcohol  until  the  volume  of  the  mixture  was  100  cc. 

A  series  of  exactly  similar  solutions  were  made  up  in  which, 
however,  25  cc.  of  N/1Q  KOH  were  employed  instead  of  25  cc. 
of  caseinate  solution. 

The  viscosities  of  these  solutions  were  determined  at  30  degrees 
in  an  Ostwald  viscometer  (5),  for  which  the  time  of  outflow,  for 
water,  was  90  seconds.  The  times  of  flow  were  read  with  a  stop 
watch.  The  viscosities  were  calculated  from  the  formula 

—  =  — - ,  where  rjo  is  the  viscosity  of  distilled  water,  t\  that  of  the 

solution  under  investigation,  s0  and  t0  are  the  density  and  the 
time  of  outflow,  respectively,  of  distilled  water  and  S  and  t  are 
the  density  and  time  of  outflow,  respectively,  of  the  solution. 
The  densities  of  the  solutions  were  determined  by  means  of  a 
normal  hydrometer,  reading  the  density  to  within  0.002. 

Taking  the  viscosity  of  water  at  30  degrees  to  be  0.00798 
dyne  per  cubic  centimeter  (13)  and  its  density  as  0.996,  the 
following  were  the  results  obtained : 

*  In  this  connection  the  work  of  H.  C.  Jones  and  collaborators  on  the  con- 
ductivities of  solutions  of  inorganic  salts  in  alcohol-water  and  acetone-water 
mixtures  should  be  consulted  (3).  Cf.  also  Walden  (14). 


VISCOSITY 


267 


TABLE  X 

Concentration  of  KOH  neutralized  to  phenolphthalein  by 
Casein  =0.0250  N 


Per  cent  of  alcohol 
by  volume 

H  in  dynes  per  cc.  for 
solvent 

H  in  dynes  per  cc.  for 
solution 

Difference  due  to 
caseinate 

0 

0.00816 

0.01668 

0.00852 

10 

0.01065 

0.02049 

0.00984 

20 

0.01376 

0.02484 

0.01108 

30 

0.01694 

0.02814 

0.01120 

40 

0.01913 

0.03237 

0.01324 

50 

0.02064 

0.03400 

0.01336 

60 

0.02011 

0.03200 

0.01189 

75 

0.01735 

0.02345 

0.00610 

Walden  (loc.  cit.)  found  that  for  solutions  of  tetraethylammo- 
nium  iodide  in  thirty  organic  solvents  the  product  of  the  molec- 
ular conductivity  of  the  salt  at  infinite  dilution  ( =  u  +  v  or, 
the  sum  of  the  migration-velocities  of  its  ions)  and  the  viscosity 
of  its  infinitely  dilute  solution  is  nearly  constant,  indicating  an 
inverse  proportionality  between  the  viscosity  of  the  solution 
(or  what  comes  to  the  same  thing  at  infinite  dilution,  the  solvent) 
and  the  migration-velocities  of  the  ions  which  it  contains.  In 
the  accompanying  table  are  given  the  values  of  p  (u  +  v)  X  f?  sol- 
vent, for  the  various  solutions  investigated. 

TABLE  XI 


Per  cent  of  alcohol 

p  («  +  f).i7  solvent 

Per  cent  of  alcohol 

p  (u  +  v).t)  solvent 

0 
10 
20 
30 

0.68X10-5 
0.69X10-5 
0.68X10-5 
0.64X10-5 

40 
50 
60 
75 

0.57X10-5 
0.50X10-6 
0.39X10-6 
0.60X10-5 

It  is  evident  that  the  product  of  the  ionic  mobility  of  potas- 
sium caseinate  ions  at  infinite  dilution  and  the  viscosity  of  the 
solvent  varies  very  much  less  than  either  of  these  quantities 
taken  alone,  indicating  in  a  qualitative  sense  at  all  events,  that 
the  ionic  mobilities  of  the  caseinate  ions  are  in  the  main  determined 
by  the  viscosity  of  the  solvent.  The  observed  increase  in  the  ionic 
mobility  of  the  caseinate  ions  on  increasing  the  alcohol  content 
of  the  solvent  from  60  to  75  per  cent,  in  view  of  the  fact  that  the 
viscosity  of  the  solvents  passes  through  a  maximum  at  from  50 


268  ELECTROCHEMISTRY 

to  60  per  cent  alcohol  and  rapidly  decreases  between  60  and 
75  per  cent  alcohol,  is  especially  confirmatory  of  this  view. 

It  will  be  noted  that  the  viscosity  of  the  solvent  is  notably 
increased  by  the  addition  of  the  concentrations  of  casein  em- 
ployed, and  this  effect,  of  course,  must  vary  very  considerably 
with  the  dilution;  yet  the  Ostwald  law  holds  good,  and  despite  the 
fact  that  the  dissolved  substance  itself  very  materially  affects  the 
viscosity  of  the  solutions,  it  is  the  viscosity  of  the  solvent  alone  which 
determines  the  mobility  of  the  protein  ions.  We  can  only  conclude 
from  this  that  that  portion  of  the  viscosity  of  the  solution  which 
is  attributable  to  the  caseinate  itself  does  not  in  any  appreciable 
degree  interfere  with  the  mobility  of  the  protein  ions.  The  signif- 
icance of  this  fact  will  be  more  fully  discussed  in  Chap.  XIII. 

6.  The  Molecular  Condition  of  Potassium  Caseinate  in  75 
Per  Cent  Alcohol.  —  We  have  seen  that  the  behavior,  optical,* 
and  electrical,  of  potassium  caseinate  dissolved  in  alcohol-water 
mixtures,  undergoes  an  abrupt  change  in  passing  from  60  to 
75  per  cent  alcohol-content.  The  degree  of  dissociation,  which 
is  but  little  affected  by  lower  concentrations  of  alcohol,  under- 
goes a  profound  diminution  in  75  per  cent  alcohol,  and  the 
opacity  of  the  solution  undergoes  a  concurrent  increase.  The 
phenomena  suggest  the  possibility  that  this  concentration  of 
alcohol  leads  not  only  to  the  formation  of  anhydrides  of  the 
protein  and  therefore,  as  explained  in  Chaps.  I  and  VI,  to  a  dimi- 
nution of  the  degree  of  dissociation,  but  also  to  a  polymerization 
of  the  protein,  as  outlined  in  equations  (i)  to  (iv)  in  Chap.  VI. 
This  polymerization  might,  very  conceivably,  be  accompanied 
by  an  increase  in  the  weight  of  casein  involved  in  the  transport 
of  one  atomic  charge.  Accordingly,  experiments  were  under- 
taken with  a  view  to  ascertaining  the  effect  of  increasing  alcohol- 
content  of  the  solvent  upon  the  electrochemical  equivalent  of 
casein. 

The  experiments  were  carried  out  in  the  manner  described 
in  Chap.  VIII,  upon  solutions  containing  50  and  75  per  cent  of 
alcohol,  made  up  in  the  manner  described  above.  The  altera- 
tions in  the  percentage  casein-content  of  the  solutions  due  to 
deposition  of  the  casein  upon  the  anode,  was  estimated  from  the 

*  Not  only  is  the  opalescence  of  the  solution  greatly  increased  but  also  the 
change  in  the  refractive  index  of  the  solvent  due  to  the  introduction  of  one 
gram  of  casein  per  100  c.c.  is  considerably  diminished  (Cf.  Chap.  XIV). 


MOLECULAR  CONDITION 


269 


change  in  the  refractive  indices  of  the  solutions,  the  proportion 
between  the  percentage  of  casein  in  these  solvents  and  the  change 
in  the  refractive  index  of  the  solvent,  due  to  the  protein,  having 
been  previously  determined  (Cf.  Chap.  XIV).  The  volume  of  the 
solution  employed  being  always  25  cc.,  the  actual  amount  of 
casein  deposited  by  the  current  was  obtained  by  dividing  the 
alteration  in  percentage  by  4. 

The  results  obtained  are  tabulated  below,  the  figures  in  the 
6th  column  being  obtained  from  those  in  the  5th  column  through 
multiplication  by  the  Faraday  constant. 

TABLE  XII* 

50  X  10-*  Equivalents  of  KOH  per  Gram  of  Casein.     Concentration  of 
KOH  neutralized  by  Casein  =  0.015  N 


Per 
cent  of 
alcohol 

Current  in 
amperes 

Time  of  passing 

Grams  casein 
lost  from  solution 

Electrochemical 
equivalent  in 
grams  per  cou- 
lomb 

Grams  of  casein 
carrying  one  ionic 
charge 

0 

2336  ±183 

50 
75 

6.88  x  10-« 
1.94  x!0-« 

3  hrs.  25  min. 
4  hrs.    0  min. 

0.2550  ±0.0170 
0.1260  ±0.0200 

0.0301  ±0.0020. 
0.0452  ±0.0072 

2906  ±193 
4363  ±695 

*  The  weight  of  casein,  in  grams,  which  carries  one  atomic  charge,  in  aqueous  solution,  is  cited 
after  the  determinations  enumerated  in  Chap.  VIII.  The  amount  of  casein  lost  from  the  anode 
through  resolution  in  electrolysis  is  not  allowed  for  in  the  above  estimates.  In  solutions  con- 
taining alcohol  it  is  probably  very  small,  since  the  rate  of  solution  of  casein  in  alkaline  solvents 
is  much  diminished  by  alcohol. 

The  smallness  of  the  current  employed  in  the  electrolysis  of  the  solution  containing  75  per  cent 
alcohol  was  due  to  the  high  resistance  of  the  caseinate  solution.  The  precipitate  from  the  solution 
containing  alcohol  is  slimy  and  not  spongy  as  it  is  from  aqueous  solution.  Nor,  in  75  per  cent 
alcohol,  does  it  adhere  well  to  the  anode  so  that  precipitation  appears  to  take  place  in  the  body  of 
the  anodal  portion  of  the  fluid,  and  the  solution  had  to  be  filtered  to  remove  the  casein  precipitated 
by  the  electrolysis. 

There  is  evidently  a  very  marked  increase  in  the  weight,  or 
decrease  in  the  valency  of  casein  ions  in  solution  of  potassium 
caseinate  when  the  added  alcohol  attains  75  per  cent,  leading 
to  a  doubling  of  the  weight  of  casein  required  to  transport  one 
atomic  charge  of  electricity. 

From  these  facts  and  from  the  failure  of  Ostwald's  dilution- 
law  to  adequately  represent  the  behavior  towards  dilution  of 
solutions  of  potassium  caseinate  in  75  per  cent  alcohol,  and  the 
opalescence  of  these  solutions,  it  appears  probable  that  they  par- 
take rather  of  the  character  of  suspensions  than  of  true  solutions, 
and  that  the  transport  of  electricity  by  the  caseinate  in  75  per 


270  ELECTROCHEMISTRY 

cent  alcohol  is  a  phenomenon  of  "electric  endosmose"  rather  than 
of  true  electrolytic  conduction  through  the  migration  of  ions. 

7.  The  Chemical  Mechanics  of  the  Coagulation  of  Proteins 
by  Alcohol.  —  We  have  seen  that  when  potassium  caseinate  is 
dissolved  in  varying  concentrations  of  alcohol,  the  opalescence  of 
the  solution  undergoes  an  abrupt  increase  in  the  neighborhood  of 
70  per  cent,  indicating  the  beginning  of  coagulation,  which, 
however,  does  not  proceed  far  enough,  even  on  further  addition 
of  alcohol,  to  lead  to  the  actual  separation  of  flocculi.  We  have 
also  seen  that  for  solutions  containing  60  per  cent  and  less  of 


alcohol  the  law  —         =  constant  holds  good  and  that  this 

Sale.  H20 

implies  that  almost  the  sole  effect  of  the  alcohol  in  these  con- 
centrations is  to  modify  the  ionic  mobility  of  the  casein  ions, 
leaving  the  degree  of  dissociation  of  the  caseinate  comparatively 
unaffected.  In  solutions  containing  75  per  cent  of  alcohol, 
however,  the  degree  of  dissociation  of  the  caseinate  is  profoundly 


diminished   and,   consequently,   the   law  —     -  =  constant    no 

Sale.  H2O 

longer  holds'  good.  On  the  basis  of  the  hypothesis  and  experi- 
mental data  outlined  in  preceding  chapters  this  phenomenon 
can  readily  be  interpreted.  We  have  seen  that  the  protein 
salts  dissociate,  not  at  terminal  —  NH2  or  —  COOH  groups  but 
at  —  COH.N—  groups  within  the  molecule.  If,  however,  the 
terminal  —  NH2  and  —COOH  groups  of  the  molecule  are  bound 
together,  as  they  are  in  anhydrides  of  the  type  HN.RCOH.NR.CO, 

|  _  I 

then  this  dissociation  can  no  longer  occur.  We  have  already 
(Chap.  VI)  seen  strong  reason  for  concluding  that  the  coagula- 
tion of  proteins  is  accomplished  by  dehydration  of  the  protein; 
the  electrochemical  behavior  of  caseinates  dissolved  in  alcohol- 
water  mixture  is  clearly  in  harmony  with  this  view. 

As  explained  in  Chap.  VI,  section  6,  equations  (i)  to  (iv),  how- 
ever, not  merely  "internal  neutralization"  of  the  protein  molecule 
may  result  from  the  dehydration  of  its  terminal  —  NH2  and 
—  COOH  groups,  but  also  polymerization.  The  high  electro- 
chemical equivalent  of  potassium  caseinate,  dissolved  in  75  per 
cent  alcohol,  and  the  opacity  of  its  solution  favor  the  view  that 
this  phenomenon  also  occurs  in  these  solutions. 

From  the  effects  of  alcohol  upon  the  rate  of  solution  of  casein 


CHEMICAL  MECHANICS  OF  COAGULATION  271 

by  alkalies  Robertson  and  Miyake  (11)  have  also  been  led  to 
infer  that  alcohol  induces  polymerization  of  the  casein  molecules 
even  in  solutions  of  sodium  caseinate  in  20  to  30  per  cent  alcohol. 
From  the  above  results,  however,  we  must  infer  that  if  in  such 
solutions  the  mass  of  the  casein  ions  is  doubled  their  valency 
must  also  be  doubled,  for  otherwise  p  would  be  halved  and  the 

observed  constancy  of  the  ratio    XH*°     in  these  solutions  would 

#H,O.alc. 

not  be  obtained. 

Incidentally,  the  profound  diminution  in  the  degree  of  disso- 
ciation of  the  caseinate  which  results  when  the  alcohol-concen- 
tration attains  a  certain  value  explains,  independently  of  any 
theory  as  to  the  mode  of  dissociation  of  the  protein  salts  in  solution, 
the  observed  fact  that  alcohol  precipitates  protein  salts  as  such 
from  their  solutions  (Cf.  Chap.  IV,  2)  and  not  the  uncombined 
proteins,  since,  previously  to  their  precipitation,  the  combined 
base  or  acid  is  bound  up  in  an  undissociated  molecule. 

LITERATURE  CITED 

(1)  Arrhenius,  S.,  Zeit.  f.  physik.  Chem.  1  (1887),  p.  285. 

(2)  Cohen,  E.,  Zeit.  f.  physik.  Chem.  25  (1898),  p.  31. 

(3)  Jones,  H.  C.,  and  Collaborators,  Amer.  Chem.  Journ.  28  (1902),  p. 

329;  32  (1904),  p.  521;  34  (1905),  p.  481;  36  (1906),  pp.  325  and 
427;  37  (1907),  p.  405;  41  (1909),  p.  433;  42  (1909),  p.  37.  Zeit.  f. 
physik.  Chem.  61  (1908),  p.  641;  62  (1908),  p.  41, 

(4)  Osborne,  W.  A.,  Journ.  Physiol.  27  (1901),  p.  398. 

(5)  Ostwald,  W.,  and  Luther,  R.,  Phys.-chem.  Messungen,  2  Aufl.  p.  260. 

(6)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.  5  (1908),  p.  147. 

(7)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  13  (1909),  p.  469. 

(8)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  14  (1910),  p.  377. 

(9)  Robertson,  T.  Brailsford,  Journ.  physical  Chem.  15  (1911),  p.  387. 

(10)  Robertson,  T.  Brailsford,  and  Burnett,  T.  C.,  Journ.  Biol.  Chem.  6 

(1909),  p.  105. 

(11)  Robertson,  T.  Brailsford,  and  Miyake,  K,  Journ.  Biol.  Chem.  26  (1916), 

p.  129. 

(12)  Roth,  W.  A.,  Zeit.  f.  physik.  Chem.  42  (1903),  p.  209. 

(13)  Thorpe,  T.  E.,  and  Rodger,  J.  W.,  Phil.  Trans.  Roy.  Soc.  London, 

185A  (1894),  p.  397. 

(14)  Walden,  P.,  Zeit.  f.  physik.  Chem.  55  (1906),  p.  209. 


PART  III 
THE  PHYSICAL  PROPERTIES  OF  PROTEIN  SYSTEMS 


CHAPTER  XII 

THE  PHENOMENA  WHICH  ACCOMPANY   CHANGES  IN  THE 
STATE   OF  AGGREGATION   OF  PROTEINS 

1.  The  Passage  of  Dry  Protein  into  Solution.  —  The  question 
whether  or  not  the  proteins  as  a  class  possess  a  definite  solubility 
in  water  is  one  which  has  not  so  far  admitted  of  any  satisfactory 
solution.  True,  certain  sparingly  soluble  salts  of  the  proteins 
undoubtedly  are  soluble  only  to  a  definite  extent  in  water,  as 
for  example  the  researches  of  Galeotti,  cited  in  Chap.  VI,  reveal; 
when  this  limit  of  solubility  is  overstepped  the  protein  salt  is 
precipitated;  then  other  forms  of  protein,  coagulated  proteins, 
" denatured"  proteins,  and  so  forth  are  definitely  insoluble  in 
water  under  the  conditions  which  accompany  their  formation.  But 
the  very  proteins  which  display  these  phenomena  under  certain 
conditions,  under  somewhat  different  conditions,  either  in  the 
form  of  free  protein  or  of  a  salt,  appear  to  be  indefinitely  soluble 
in  water;  miscible  with  it  in  all  proportions.  The  phenomena 
of  "  solution,"  chemical  combination  with  water,  and  of  the 
hygroscopic,  possibly  purely  physical,  retention  of  water,  become, 
in  these  cases  and  so  far  as  our  knowledge  at  present  extends, 
inextricably  confused. 

On  the  whole  it  appears  very  probable  that  such  proteins  and 
protein  salts  as  gelatin  and  the  caseinates  are  not  only  miscible 
with  water  in  all  proportions,  but  also  capable  of  entering  into 
chemical  combination  with  water  in  many  different  proportions. 
To  this  view  Pauli  (72),  in  contradistinction  to  Hardy  (34)  (35), 
also  inclines. 

Base-  and  acid-free  casein  is  definitely  insoluble  in  distilled 
water;  but  in  water  containing  a  base  its  solubility  would  appear 
to  be  limited  only  by  the  quantity  of  base  which  the  water  con- 
tains and  not  by  the  volume  of  water.  The  casein  continues 
to  dissolve  until,  as  explained  in  Chaps.  V  and  X,  11.4  X  10~5 
equivalents  of  base  are  combined  with  each  gram  of  casein  and 
solution  of  the  casein  stops  merely  because  the  power  of  the  base 
to  neutralize  casein  is  exhausted,  and  not  because  the  solvent 

275 


276  PHYSICAL  PROPERTIES 

will  hold  no  more  of  the  caseinate.  Addition  of  alkali  to  the 
mixture  results  in  the  passage  of  more  casein  into  solution; 
addition  of  acid  in  the  throwing  of  uncombined  casein  out  of 
solution.  Apparently  casemates,  of  the  alkalies  at  all  events, 
are  indefinitely  soluble  in  water,  although  in  practice  we  are 
limited  in  the  preparation  of  strong  solutions,  not  only  of  the 
caseinates  but  of  other  proteins,  by  the  viscous  and  glairy  nature 
of  such  solutions,  and  the  consequent  difficulty  of  ensuring  their 
homogeneous  character.  Corresponding  to  this  fact  we  find 
that  it  is  very  difficult  to  prepare  the  caseinates  of  the  alkalies 
in  the  form  of  dry  powders.  I  have  precipitated  lithium  casein- 
ate  from  90  per  cent  alcohol  by  the  addition  of  ether  and  en- 
deavored to  dry  this  product  by  washing  with  ether  and  standing 
over  sulphuric  acid  at  30  degrees.  The  result  is  merely  a  soft 
gelatinous  mass;  on  prolonged  standing  over  sulphuric  acid 
(a  matter  of  weeks)  it  becomes  horny;  at  no  definite  time  can 
we  state  that  the  product  has  become  anhydrous;  it  simply 
exhibits  a  continual  change  in  consistency  with  the  continual 
withdrawal  of  water,  just  as  gelatin  does.  On  the  other  hand, 
proteins  which  are  insoluble  in  water  and  certain  other  proteins 
and  protein  salts  are  readily  rendered  anhydrous  by  treatment 
with  alcohol  and  can  be  prepared  through  this  means  in  the 
form  of  dry  powders  (Cf.  Chap.  II).  The  affinity  of  a  pro- 
tein for  water,  therefore,  is  a  function  which  varies,  not  only 
with  the  character  of  the  protein,  but  with  the  character  of  any 
base  or  acid  which  may  be  combined  with  it.  In  all  cases  it 
would  appear  to  be  of  considerable  magnitude,  however,  the 
difficulty  which  histologists  experience  in  thoroughly  dehy- 
drating tissues,  for  example,  being  eloquent  testimony  of  this 
fact. 

It  has  been  shown  by  Noyes  and  Whitney  (61)  (10)  that  under 
conditions  involving  no  chemical  interaction  (except  the  pos- 
sible formation  of  "solvates"  (Cf.  Chap.  VI)),  the  rate  of  solution 
of  a  crystalloid  in  water  is  at  each  moment  proportional  to  the 
difference  between  its  concentration  at  that  moment  and  its 
concentration  at  saturation  of  the  solvent.  They  conclude  from 
this  that  at  the  boundary  of  the  crystals  and  the  solution  there 
exists  a  film  of  solution,  which  is  always  saturated  and,  for  a 
constant  rate  of  stirring,  of  constant  thickness.  The  velocity 
of  solution  is  thus  determined  by  the  rate  of  diffusion  of  the 


PASSAGE  INTO  SOLUTION  277 

dissolved  crystalloid  out  of  this  film  into  the  body  of  the  fluid. 
This  leads  to  the  equation: 

log-^_  =  SA*,  (i) 

where  a  is  the  concentration  of  a  saturated  solution  of  the  crys- 
talloid, x  the  concentration  of  the  solution  at  time  t,  s  the  area 
of  the  surface  of  the  crystalloid  which  is  exposed  to  the  action 
of  the  solvent  and  k  a  constant  which  varies  with  the  rate  of 
stirring  and  with  the  rate  of  diffusion  of  the  crystalloid  (11), 
and,  consequently,  with  the  temperature. 

On  the  other  hand,  Boguski  (4)  (5)  and  Veley  (104)  (105) 
have  shown  that  the  rate  of  solution  of  basic  substances  such 
as  marble  or  zinc  in  acids,  a  process  involving  chemical  inter- 
action, is  proportional  at  each  instant  to  the  concentration  of 
the  unneutralized  acid  and  to  the  surface  of  the  solid.  Upon 
the  supposition  that  the  surface  remains  appreciably  constant 
in  area  during  the  period  occupied  by  the  experiment  (a  readily 
realizable  condition)  this  leads  to  the  equation : 

log  -r^--  =  fat,  (ii) 

Cl  ~~  •*/ 

which  is  identical  with  the  above,  save  that  a  is,  in  this  case, 
the  initial  concentration  of  the  acid.  In  this  case  also  Nernst 
believes  (60)  the  velocity  of  solution  is  determined  by  a  diffusion- 
velocity,  namely  the  velocity  with  which  the  acid  diffuses  into 
the  film  in  contact  with  the  solid.  Naturally,  were  the  velocity 
of  the  chemical  interaction  low  in  comparison  with  the  velocity 
of  diffusion  of  the  acid  (or  any  reagent  playing  a  similar  part) 
the  determining  velocity  would  be  a  chemical  and  not  a  diffusion 
velocity.  An  examination  of  this  case  in  the  light  of  the  mass 
law  shows,  however,  that  the  form  of  the  relation  between  time 
and  mass  dissolved  would  be  the  same  as  that  expressed  in 
equation  (ii).* 

In  the  light  of  what  has  been  said  concerning  the  indefinite 
solubility  of  the  caseinates  in  water,  it  might  be  anticipated 
that  equation  (i)  would  not  apply  to  the  dissolving  of  solid  casein 
in  alkaline  water.  It  is  quite  imaginable,  however,  that  equa- 
tion (ii)  might  apply,  a,  in  that  equation,  being  the  number  of 

*  Provided  only  one  molecule  of  acid  (or  other  dissolved  reagent)  took  part 
in  the  reaction. 


278  PHYSICAL  PROPERTIES 

equivalents  of  base  in  the  water  employed  to  dissolve  the  casein. 
The  experimental  fact  is,  however,  that  neither  of  these  equations 
expresses  the  relationship  between  the  time  for  which  a  mixture 
of  solid  casein  and  alkaline  water  is  stirred  and  the  mass  of 
casein  dissolved  (86). . 

In  the  experiments  about  to  be  described  the  casein  which 
was  employed  was  especially  prepared  so  as  to  readily  sink  in 
water  (Cf.  Chap.  II,  2),  that  is,  it  was  not  absolutely  anhydrous 
but  contained  a  trace  of  water  insufficient  to  disturb  the  accu- 
racy of  the  results,  but  very  material  to  the  success  of  the  ex- 
periments. Since  the  presence  and  amount  of  associated  traces 
of  water  play  such  an  important  part  in  determining  the  readi- 
ness with  which  casein  is  wetted  by  a  solvent,  it  proved  to  be 
necessary,  in  obtaining  comparable  experimental  results,  always 
to  employ  the  same  preparation  of  casein,  since  no  two  samples 
of  incompletely  anhydrous  casein  can  be  relied  upon  to  contain 
exactly  the  same  proportion  of  water. 

A  measured  amount  (usually  100  cc.)  of  the  fluid  employed 
as  solvent  was  placed  in  a  beaker  of  squat  form  and  400  cc. 
capacity  and  was  agitated  by  a  flattened  glass  rod  which  was 
bent  at  right  angles,  the  plane  of  the  horizontal  arm  being  some- 
what inclined  to  the  vertical,  so  as  to  communicate  an  upward 
thrust  to  the  rotating  liquid.  The  horizontal  arm  of  the  stirrer 
was  about  2|  cm.  long  and  as  near  as  possible  to  the  bottom  of 
the  beaker;  this  was  rotated  at  an  approximately  constant  rate 
of  about  1600  revolutions  per  minute  by  a  small  motor.  While 
stirring,  a  weighed  amount  of  the  casein  was  dropped  into  the 
fluid.  At  stated  intervals  samples  of  the  mixture  were  almost 
instantaneously  abstracted  by  means  of  a  10  cc.  pipette  which 
was  provided  with  a  rubber  bulb.  The  samples  were  then  very 
rapidly  filtered  through  lightly  packed  glass  wool.  The  time 
occupied  in  filtration  was  never  more  than  30  seconds  for  the 
5  and  10  minute  samples,  or  more  than  1  minute  for  the  later 
samples.  The  refractive  index  of  the  filtrate  from  each  sample 
was  then  determined.  Denoting  the  refractive  index  of  any 
given  sample  by  n  and  that  of  the  pure  solvent  by  wi,  the  quotient 
n  ~  Ul  is  the  number  of  grams  of  casein  dissolved  in  100  cc. 

U.UUlO^w 

of  the  solvent  at  the  moment  when  the  sample  was  extracted 
(Cf.  Chap.  XIV). 


PASSAGE  INTO  SOLUTION 


279 


The  type  of  relation  which  was  found  to  subsist  between  the 
time  which  elapsed  after  the  introduction  of  the  casein  and  the 
number  of  grams  of  casein  dissolved  in  100  cc.  of  solvent  is  shown 
diagrammatically  in  the  accompanying  figure,  in  which  the 
abscissae  represent  minutes  and  the  ordinates  the  number  of 
grams  of  casein  dissolved  in  100  cc.  of  solvent.  It  will  be  ob- 
served that  the  rate  of  solution  is  at  first  very  great,  but  that 
it  very  rapidly  falls  off.  It  does  not  fall  to  zero,  however,  that 
is,  the  curve  does  not  appear  to  approach  an  asymptote,  but 
is  rather  of  a  parabolic  form.  Nor  does  this  appear  strange 
when  we  observe  that  although,  after  two  hours  of  stirring,  the 


Minutes 


rate  of  solution  of  the  casein  is  very  small,  yet  the  solvent  is 
still  very  far  from  being  " saturated"  with  casein.  The  alkali- 
equivalent  of  1  gram  of  casein  is,  as  we  have  seen  (Chaps.  V, 
IX  and  X),  11.4  X  10~5  equivalent  gram  molecules,  but  the 
proportion  of  the  casein  actually  dissolved  to  the  amount  of 
base  present  in  the  solvent  was  always,  even  after  two  hours 
of  stirring  at  room  temperatures,  very  much  less  than  this.* 

*  It  might  be  imagined  that  the  solutions  of  the  casemates  which  contain 
only  11.4  X  10~5  equivalents  of  base  per  gram  of  casein,  and  which  are  pre- 
pared by  neutralizing  the  excess  of  base  which  is  employed  to  dissolve  the 
casein  by  the  addition  of  a  strong  acid,  are  "supersaturated"  with  respect 
to  casein.  Two  facts  speak  very  strongly  against  this  view,  however.  The 
first  is  that  the  electro-chemical  behavior  of  casein  in  much  more  alkaline 
solutions  already  foreshadows  the  fact  that  the  least  proportion  of  alkali 
which  will  hold  one  gram  of  casein  in  solution  is  11.4  X  10~5  equivalents  (Cf. 
Chap.  X).  The  second  is  that  if  a  3  per  cent  solution  of  casein  in  0.0043 
N  KOH  (1  gram  of  casein  to  14  X  10~5  equivalents  of  base)  be  prepared  by 
dissolving  casein  hi  excess  of  base  and  then  neutralizing  the  excess  with  acid, 
it  can  be  kept  in  a  sealed  glass  vessel,  in  the  presence  of  excess  of  toluol  for 
6  months  without  any  deposition  of  casein  occurring.  At  the  end  of  from 
10  months  to  a  year  a  very  bulky  white  precipitate  is  deposited  leaving  the 


280  PHYSICAL  PROPERTIES 

The  relation  between  the  time  of  stirring  and  the  quantity 
of  casein  dissolved  does  not  obey  either  of  the  above  cited  equa- 
tions; it  does  not  appear  to  obey  any  of  the  ordinary  chemical 
reaction  or  solution  velocity  formulae.  The  rate  at  which  the 
velocity  of  solution  falls  off;  the  negative  acceleration  of  the 
process;  is  far  too  great  to  permit  of  representation  by  either 
of  the  formulae  (i)  and  (ii).  Nor  is  any  better  agreement  ob- 
tained if  we  insert,  for  the  value  of  a  in  these  equations,  the 
actual  number  of  grams  of  casein  present  in  the  mixture,  or  if, 
allowing  for  the  diminution  in  the  surface  of  the  casein  exposed 
to  the  action  of  the  solvent,  as  solution  proceeds,  we  endeavor 
to  apply  the  relation 

g  =  K(A  -  x)  (B  -  x),  (iii)* 

where  A  is  the  number  of  grams  of  casein  which  the  amount  of 
alkali  present  in  the  solvent  is  capable  of  holding  in  solution, 
B  is  the  number  of  grams  of  casein  actually  present  in  the  mix- 
ture, x  is  the  amount  of  casein  dissolved  at  any  given  moment 
and  K  is  a  constant.  The  relation  between  the  time  of  stirring 
and  the  amount  of  casein  dissolved,  however,  does  obey,  very 
accurately,  the  relation 

x  =  Ktm,  (iv) 

where  x  is  the  amount  dissolved  after  time  t,  and  K  and  m  are 
constants  which  vary  with  the  nature  and  concentration  of  the 
alkali  solution  employed  as  solvent  and  with  the  total  mass  of 
casein  present  in  the  mixture. 
The    following    tables    enumerate    illustrative    results,  f    The 

supernatant  fluid  very  nearly  clear.  It  must  be  recollected,  however,  that 
when  periods  of  time  of  the  order  of  a  year  are  concerned  no  protein  solution 
can  be  regarded  as  being  in  equilibrium.  Even  in  absolutely  neutral  solutions 
the  H+and  OH'  ions  of  the  water  lead  to  a  measure  of  "autohydrolysis"  (Cf. 
Chap.  XVI)  and  the  above  solution  was  distinctly  acid  (Cf.  Chaps.  V  and  IX). 
Hydrolysis  of  caseinates  of  the  bases  leads  to  the  formation  of  paranucleins 
which  are  insoluble  in  neutral  and  faintly  acid  solutions,  and  to  substances 
which  bind  bases  and  therefore  tend  to  abstract  them  from  the  casein  (Cf. 
Chap.  XVI)  and  consequently,  when  the  total  amount  of  base  present  is  so 
very  little  more  than  enough  to  hold  the  casein  in  solution,  to  indirectly  pre- 
cipitate the  casein  itself. 

*  In  its  integrated  form:  log^(/l  ~  X\   =  Kt. 

A  (n  —  X) 

t  For  additional  experimental  results  the  reader  is  referred  to  my  original 
communication  (86). 


PASSAGE  INTO  SOLUTION 


281 


TABLE  I 

Solvent:  0.00870  N  KOH.    Temperature  18-20  degrees 
#  =  1.48        m  =  0.146 


Time  in  minutes 

Grams  casein  dissolved  in  100  cc.  solvent 

A 

Found 

Calculated 

5 
10 
30 
60 
120 

1.84 
2.11 
2.43 
2.70 
2.96 

1.87 

2.07 
2.43 
2.69 
2.98 

+0.03 
-0.04 
±0.00 
-0.01 
+0.02 

SA  =  ±0.00 

TABLE  II 

Solvent:  0.00870  N  NaOH.    Temperature  22-23 
K  =  1.33        m  =  0.163 


Time  in  minutes 

Grams  casein  dissolved  in  100  cc.  solvent 

A 

Found 

Calculated 

5 

10 
30 
60 
120 

1.71 
1.97 
2.30 
2.63 
2.89 

1.73 
1.94 
2.32 
2.60 
2.91 

+0.02 
-0.03 
+0.02 
-0.03 
+0.02 

SA  =  ±0.00 

TABLE  III 

Solvent:  0.01740  N  Ba(OH)2.    Temperature  20  degrees 
K  =  1.34        m  =  0.194 


Time  in  minutes 

Grams  casein  dissolved  in  100  cc.  solvent 

A 

Found 

Calculated 

5 
10 
30 
60 
120 

1.85 
2.05 
2.64 
3.03 
3.36 

1.84 
2.10 
2.60 
2.98 
3.40 

-0.01 
+0.05 
-0.04 
-0.05 
+0.04 

SA  =  -0.01 

282  PHYSICAL  PROPERTIES 

quantity  of  solvent  employed  was  100  cc.  and  the  number  of 
grams  of  casein  initially  added  to  it  was  5.  For  a  reason  which 
will  shortly  appear  no  especial  effort  was  made  to  maintain  a 
constant  temperature  during  the  progress  of  an  experiment,  but 
at  the  head  of  each  table  are  given  the  temperatures  of  the  mix- 
ture at  the  beginning  and  at  the  end  of  the  experiment.  The 
temperature  at  the  beginning  of  the  experiment  is  placed  first. 
In  the  column  headed  " calculated"  are  given  the  values  of  x 
calculated  from  the  above  formula,  the  constants  K  and  m  being 
determined  from  all  of  the  observations  by  the  method  of  least 
squares,  employing  for  this  purpose  the  form: 

logio  x  =  m  logio  t  +  logio  K. 

The  possible  experimental  error  in  the  determination  of  the 
concentration  of  a  casein  solution  by  means  of  its  refractive 
index  is  always  ±0.07  gram  per  100  cc.  It  will  be  seen  that  the 
differences  (=  A)  between  the  observed  and  calculated  values  of 
x  are  usually  considerably  less  than  the  possible  error  in  the 
determination  of  the  concentration  of  the  casein  in  the  filtrates. 

Equally  concentrated  solutions  of  KOH,  NaOH,  and  NH4OH 
dissolve  casein  with  about  equal  rapidity,  while  solutions  of  the 
hydroxides  of  the  alkaline  earths  dissolve  casein  much  more 
slowly,  Sr(OH)2  dissolving  the  casein  most  rapidly,  Ca(OH)2 
more  slowly  and  Ba(OH)2  more  slowly  still.  As  I  have  pointed 
out  in  Chaps.  X  and  XI  this  fact  is  of  significance  when  viewed 
in  the  light  of  the  facts  that  solutions  of  the  caseinates  of  the 
alkaline  earths  become  opalescent  on  heating,  while  those  of  the 
caseinates  of  the  alkalies  and  ammonium  do  not  (63),  that  the 
caseinates  of  the  alkaline  earths  will  not  pass  through  the  pores 
of  a  clay  filter,  while  those  of  the  alkalies  and  ammonium  readily 
do  so  (63),  and  that  in  these  and  in  other  ways  the  caseinates 
of  the  alkaline  earths  give  evidence  of  being  present,  in  their 
solutions,  in  the  form  of  more  bulky  molecules  than  those  of 
the  caseinates  of  the  alkalies  and  of  ammonium  under  equivalent 
conditions.  It  appears  probable  that  when  casein  is  suspended 
in  solutions  of  the  hydroxides  of  the  alkaline  earths  the  soluble 
caseinate  is  just  as  readily  formed  as  it  is  in  solutions  of  the 
alkalies,  but  that  it  is  hindered  in  passing  out  of  the  casein  par- 
ticles, through  their  capillary  pores,  into  the  solution,  just  as  it 
is  hindered  in  passing  through  the  pores  of  a  clay  filter.  We 


PASSAGE  INTO  SOLUTION 


283 


have  had  occasion  to  comment  upon  the  fact  (Cf.  Chap.  V) 
that  dry  casein  will  not  pass  into  solution  in  dilute  acids,  although 
wet,  flocculent,  freshly  precipitated  casein  will  do  so;  never- 
theless, acid  is  taken  up  from  the  solution  and  bound  by  the 
casein  (103).  It  appears  probable,  as  I  indicated  in  Chap.  V, 
that  in  this  case  soluble  casein  salts  are  formed  but  that  they 
are  prevented,  by  the  grossness  of  their  molecules,  from  passing 
out  through  the  pores  of  the  casein  particles  into  the  body  of 
the  solvent. 

The  influence  of  neutral  salts  upon  the  rate  of  solution  of 
casein  by  dilute  sodium  hydroxide  has  been  investigated  by 
Robertson  and  Miyake  (89)  who  find  that  even  in  the  presence 
of  high  concentrations  of  these  salts  the  relationship  between 
the  time  and  the  amount  of  casein  dissolved  is  expressed  by  the 
relation  x  =  Ktm.  The  presence  of  the  salts  retards  the  solu- 
tion of  the  casein,  however,  the  retardation  due  to  alkaline 
earth  chlorides  being  about  one  hundred  times  as  great  as  that 
which  is  brought  about  by  chlorides  of  the  alkalies.  The  degree 
of  retardation  increases  with  increase  of  the  concentration  of 
salt  employed. 

The  amount  of  casein  which  is  dissolved,  in  a  given  period  of 
time,  by  a  solution  of  KOH  is,  within  the  limits  of  accuracy  of 
the  determinations,  directly  proportional  to  the  concentration  of 
the  KOH  solution.  This  is  very  clearly  shown  in  Table  IV,  in 
which  r  denotes  the  ratio  of  the  number  of  grams  of  casein  dis- 
solved to  the  number  of  equivalent  gram  molecules  (multiplied 
by  100)  of  KOH  present  in  100  cc.  of  solvent  employed.  It  will 


TABLE  IV 


Concentration  of  the  KOH  solution  employed  as  solvent 

Time  in 

minutes 

0.  00218  N 

0.00435  N 

0.00653  N 

0.  00870  tf 

0.01088  N 

0.  01305  N 

0.  01523  N 

0.01740  AT 

r 

r 

r 

r* 

r 

r 

r 

r 

5 

21 

18 

16 

20 

19 

21 

20 

19 

10 

23 

21 

18 

23 

23 

24 

22 

21 

30 

29 

26 

26 

28 

27 

28 

26 

26 

60 

33 

29 

29 

31 

30 

31 

28 

28 

120 

33 

31 

31 

34 

33 

33 

30 

*  Average  of  two  determinations  at  26.5  degrees  and  18  to  20  degrees,  respectively. 


284  PHYSICAL  PROPERTIES 

be  observed  that  this  ratio,  for  any  of  the  given  periods  of  time, 
is  very  nearly  constant.* 

The  temperature-coefficient  of  the  velocity  of  solution  is  very 
small.  The  difference  between  the  amounts  of  casein  dissolved, 
after  a  given  time,  at  18-20  degrees  and  at  26  degrees  are  only 
slightly  greater  than  or  equal  to  the  possible  error  of  the  deter- 
minations. So  far  as  the  accuracy  of  the  method  employed 
enables  us  to  decide,  the  temperature-coefficient  of  the  rate  of 
solution,  between  the  temperatures  of  20  and  36  degrees,  is  prac- 
tically zero.f  At  higher  temperatures  the  rate  of  solution  in 
solutions  of  the  hydroxides  of  the  alkalies  is  increased  and  the 
rate  of  solution  in  solutions  of  the  hydroxides  of  the  alkaline 
earths  is  very  materially  diminished.  At  these  temperatures, 
also,  solutions  of  the  caseinates  of  the  alkaline  earths,  which  are 
neutral  to  phenolphthalein,  become  alkaline  to  phenolphthalein, 
while  solutions  of  the  caseinates  of  the  alkalies  which  are  neutral 
to  phenolphthalein  do  not  (63).  I  have  sought  to  account  for 
these  facts  by  supposing  that  temperatures  above  36  degrees 
lead  to  a  polymerization  of  the  protein  moiety  of  the  caseinates 
of  the  alkaline  earths  (83). 

The  low  temperature-coefficient  would  in  itself  lead  us  to 
suspect  that  the  process  which  determines  the  rate  of  solution 
of  casein  in  solutions  of  bases  is  not  chemical  in  nature. 

That  the  rate  of  solution  of  the  casein  is  not  determined  by 
the  velocity  of  a  chemical  reaction  occurring  exclusively  in  the 
liquid  phase  is  also  shown  by  the  fact  that  the  rate  of  solution 
of  the  casein  is  dependent  upon  the  mass  of  casein  initially  in- 
troduced into  the  mixture.  Were  the  rate  of  solution  of  the 
casein  dependent  solely  upon  the  velocity  of  a  reaction  between 
casein  and  the  alkali,  taking  place  in  the  liquid  phase,  then  since, 
in  the  presence  of  undissolved  casein,  the  liquid  would  always 
be  saturated  with  casein,  the  rate  of  solution  should  be  inde- 
pendent of  the  mass  of  undissolved  casein.  We  are  led  to  con- 
clude, therefore,  that  the  processes  which  determine  the  velocity 
of  solution  occur,  in  part  at  all  events,  either  within  or  at  the 
surfaces  of  the  suspended  particles  of  undissolved  casein. 

*  At  "saturation"  of  the  alkali  with  casein  the  numerical  value  of  this 
ratio  would  be  91. 

t  It  was  for  this  reason,  of  course,  that  no  special  effort  was  made  to  main- 
tain a  constant  temperature  during  the  progress  of  the  experiments. 


PASSAGE  INTO  SOLUTION 


285 


TABLE  V 


Grams  casein 

added  to  200  cc. 
of  solvent 

2.5 

5.0 

7.5 

10.0 

12.5 

15.0 

(0.010  N  KOH) 

Time  in 

Grams 

Grams 

Grams 

Grams 

Grams 

Grams 

minutes 

dissolved 

dissolved 

dissolved 

dissolved 

dissolved 

dissolved 

5 

2.24 

2.76 

3.28 

3.68 

3.68 

3.82 

10 

2.36 

3.28 

3.82 

4.22 

4.22 

4.60 

30 

(2.50)* 

3.96 

4.86 

5.40 

5.40 

5.66 

60 

4.60 

5.40 

5.92 

5.92 

6.18 

*  That  is  to  say,  at  some  undetermined  time  previous  to  this  all  of  the  casein  introduced  into 
the  solvent  had  been  dissolved. 

The  relation  between  the  amount  of  casein  dissolved  in  a  given 
time  and  the  mass  of  casein  initially  added  to  the  solvent  is 
shown  in  Table  V  and  graphically  in  accompanying  figure.  The 
temperature  of  the  mixtures  was,  in  all  of  these  experiments, 


6Q  infantes 


Grams  Casein  in  Mixture 


20  degrees.  It  will  be  seen  that  the  rate  of  solution"  increases, 
at  first  somewhat  rapidly  with  the  mass  of  casein  added  to  the 
solvent,  later  more  slowly. 

Reverting  to  equation  (iv),  the  relation  x  =  Ktm  is,  it  is  of 
interest  to  observe,  the  same  as  that  found  by  Cameron  and 
Bell  (14)  (15)  and  later  confirmed  by  Ostwald  from  the  investi- 
gations of  Goppelsroeder  (66),  to  subsist  between  the  amount 
of  fluid  absorbed  by  a  column  of  sand  or  a  strip  of  filter-paper 
and  the  time  during  which  the  fluid  has  remained  in  contact 
with  a  portion  of  its  surface.* 

The  values  of  the  constants  are  also  of  the  same  order  of  mag- 
nitude as  those  found  in  these  investigations.  It  is  possible, 
therefore,  that  the  rate  of  solution  of  the  casein  is  primarily 

*  According  to  Cameron  and  Bell  (14),  this  formula  can  be  derived  from 
the  formula  of  Poiseuille  for  the  flow  of  liquids  through  capillary  spaces. 


286  PHYSICAL  PROPERTIES 

determined  by  the  rate  at  which  the  particles  are  penetrated 
and  wetted  by  the  solvent,  the  process  of  chemical  reaction 
between  the  alkali  and  the  casein  taking  place  at  a  relatively 
great  velocity. 

It  is,  perhaps,  not  surprising  that  the  factor  which  determines 
the  rate  of  solution  of  casein  should  be  the  velocity  with  which 
it  is  wetted  by  the  solvent,  while  that  which  determines  the  rate 
of  solution  for  a  crystalloid  is  the  velocity  with  which  the  dis- 
solved substance  diffuses  out  of  a  thin  layer  of  saturated  solu- 
tion in  immediate  contact  with  the  surfaces  of  the  crystals.  A 
crystal  is  only  wetted  upon  its  external  surface,  and  the  wetting, 
naturally,  takes  place  instantly.  A  particle  of  casein  (or,  in 
general,  of  the  solid  phase  of  any  colloid)  is,  however,  comparable 
in  structure  with  a  sponge;  the  surface  which  may  be  wetted  by 
the  solvent  is,  per  unit  volume,  very  much  larger  than  that  of 
a  crystal,  and  the  solvent  must,  in  wetting  this  surface,  traverse 
a  relatively  immense  network  of  minute  capillary  pores.  Under 
such  conditions  the  time  occupied  in  wetting  the  surfaces  of  the 
particles  may  well  be  great  compared  with  the  time  required  for 
the  dissolved  substance  to  diffuse  from  these  surfaces  into  the 
solvent,  or  with  the  time  required  for  the  accomplishment  of 
the  union  between  the  protein  and  the  alkali  in  the  solvent. 

As  might  be  expected,  similar  relationships  are  encountered  in 
the  extraction  of  a  protein  from  desiccated  and  finely  divided 
fragments  of  tissue.  The  rate  of  extraction  of  the  protamin 
salmin  by  dilute  hydrochloric  acid  from  the  dried  spermatozoa 
of  the  salmon  is  determined  primarily  by  capillary  forces  (87) 
(88).  The  accompanying  chemical  phenomena  (decomposition 
of  compounds  of  salmin  within  the  tissue,  formation  of  salmin 
hydrochloride,  etc.)  occur  at  a  relatively  very  great  velocity  and 
hence  do  not  affect  the  rate  of  extraction. 

The  rate  of  extraction  or  passage  of  a  protein  from  a  colloidal 
phase  into  a  surrounding  solvent  may  be  determined  either  by 
the  rate  of  passage  of  the  soluble  protein  compound  from  within 
the  colloid  particles  into  the  surrounding  solvent,  or  by  the  rate 
of  penetration  of  the  solvent  into  the  colloid  particles,  or  by 
both  of  these  processes.  •  That  the  latter  process,  namely  the 
penetration  of  the  colloid,  is  of  very  great  importance  in  deter- 
mining the  observed  time-relations  may  be  inferred  from  the 
fact  that  the  absorption  of  acid  from  dilute  acid  solutions  by 


PASSAGE  INTO  SOLUTION  287 

suspended  particles  of  casein  takes  place  in  accordance  with  the 
equation  x  =  Ktm,  although  in  this  case  either  no  soluble  com- 
pound of  casein  is  formed  or,  which  is  more  probable,  the  soluble 
compound  which  is  formed  is  unable  to  issue  forth  from  the 
colloid  particles  within  which  it  arises  (88)  (89). 
Differentiating  the  equation: 

x  =  Kt" 
we  find 


in  other  words,  the  product  Km,  which  has  been  termed  by 
Robertson  and  Miyake  the  coefficient  of  penetration,  expresses 
the  constant  proportionality  between  the  velocity  of  solution  and 
an  exponent  (peculiar  to  each  solvent)  of  the  time  during  which 
the  protein  has  been  exposed  to  the  action  of  the  solvent.  The 
coefficient  of  penetration  is,  in  the  case  of  the  solution  of  casein 
by  dilute  sodium  hydroxide,  very  characteristically  affected  by 
the  presence  in  the  solvent  of  inorganic  salts.  The  magnitude 
of  the  coefficient  of  penetration  decreases  with  increasing  con- 
centrations of  NaCl,  KC1,  CaCl2,  SrCl2  or  BaCl2,  the  acceleration 
of  the  decrease  being  positive  in  the  cases  of  NaCl  and  KC1  and 
negative  in  the  cases  of  CaCl2,  SrCl2  or  BaCl2.  Lithium  chloride, 
on  the  other  hand,  increases  the  value  of  the  coefficient  of  pene- 
tration with  a  negative  acceleration,  so  that  at  concentrations 
of  this  salt  lying  above  0.33  normal  the  algebraic  sum  of  these 
two  opposite  effects  results  in  a  decrease  of  the  value  of  the 
coefficient  of  penetration  (89). 

If  the  rate  of  solution  of  casein  by  alkalies  were  primarily 
determined  by  the  rate  of  penetration  of  the  capillary  pores  in 
the  suspended  colloid  particles  by  the  solvent,  then  the  addition 
to  the  solvent  of  any  substance  which  markedly  reduces  the 
tension  of  a  solid-water  interface  should  retard  the  rate  of 
penetration  and,  consequently,  the  rate  of  solution  of  the  casein. 
It  has  been  found  by  Robertson  and  Miyake  (90)  that  alcohol 
and  glycerol,  both  of  which  reduce  the  tension  of  a  solid-water 
interface,  also  retard  the  solution  of  casein  by  dilute  sodium 
hydroxide.  The  penetration-formula  x  —  Ktm  expresses  the  re- 
lationship between  the  quantity  of  casein  dissolved  and  the 
time  of  stirring  of  the  mixture  in  all  mixtures  of  glycerol  and  water 


288  PHYSICAL  PROPERTIES 

and  in  alcohol-water  mixtures  which  contain  less  than  4.5  mol.  or 
more  than  7  mol.  of  alcohol.  The  formula  most  decidedly  fails 
to  apply,  however,  to  the  rate  of  solution  of  casein  in  dilute  alkali 
containing  between  4  and  8  mol.  of  alcohol,  for  in  these  mix- 
tures the  extremely  curious  phenomenon  is  observed  of  partial  re- 
precipitation,  on  continued  stirring,  of  the  casein  which  is  initially 
dissolved,  so  that  after  2  hours  of  stirring,  the  casein  dissolved 
in  these  mixtures  may  actually  be  considerably  less  than  after 
only  I  hour  of  stirring.  The  penetration-formula  obviously  im- 
plies a  continuous  and  irreversible  process,  while  in  the  presence 
of  the  above-mentioned  concentrations  of  alcohol  the  process  of 
solution  is  evidently  partially  reversible.  Above  and  below  this 
critical  zone  of  concentrations  the  relation  between  the  time  of 
stirring  and  the  concentration  of  casein  dissolved  is  adequately 
expressed  by  the  penetration-formula. 

On  examining  the  effect  of  alcohol  upon  the  magnitude  of  the 
coefficient  of  penetration  it  becomes  evident  that  in  the  zone  of 

Coefficient  of  Penetration 


0.3 


0.2 


\ 


Concentration  of 
Alcohol 


0.5    1  2  3  4  5  6  7  8  9          10         11         12         13          14  M. 

concentrations  lying  between  4  and  8  mol.  the  character  of  the 
relationship  between  the  concentration  of  alcohol  and  the  mag- 
nitude of  the  coefficient  of  penetration  is  undergoing  a  transition. 
For  concentrations  of  alcohol  lying  below  4.5  mol.,  the  coefficient 
of  penetration  decreases  with  negative  acceleration  as  the  con- 
centration of  alcohol  increases.  In  mixtures  containing  over 
7  mol.  alcohol,  however,  the  coefficient  of  penetration  decreases 
with  positive  acceleration,  as  the  concentration  of  alcohol  in- 


PASSAGE  INTO  SOLUTION  289 

creases.  In  mixtures  containing  concentrations  of  alcohol  lying 
between  4.5  and  7  mol.  inclusive,  the  character  of  the  relation- 
ship is  indeterminate.  These  relationships  are  illustrated  by 
the  accompanying  figure  in  which  the  broken  lines  indicate  the 
calculated  continuation  of  the  continuous  lines  drawn  through 
the  experimentally  determined  points,  the  formula  used  for  the 
calculated  exterpolations  being: 

XiMi  -  KM  =  ac  +  0C2, 

in  which  KiMi  —  KM  represents  the  decrease  in  the  coefficient 
of  penetration,  c  represents  the  concentration  of  alcohol,  a  is  a 
constant  which  is  positive  when  c  lies  below  4.5  mol.  and  nega- 
tive when  c  lies  above  8  mol.,  and  /?  is  a  constant  which  is  nega- 
tive when  c  lies  below  4.5  mol.  and  positive  when  c  lies  above 
8  mol. 

The  only  explanation  of  these  curious  phenomena  which  offers 
itself  lies  in  the  fact  that  alcohol  is  a  coagulant  of  proteins,  and 
therefore  brings  about  dehydration  and  consequent  polymeri- 
zation of  protein  molecules.  At  low  concentrations  of  alcohol 
we  have  to  deal  with  the  rate  of  solution  of  " single"  molecules 
of  sodium  caseinate,  at  higher  concentrations  with  the  rate  of 
solution  of  polymerized  molecules  which  we  may  for  the  sake  of 
brevity  term  "double"  molecules,  while  at  intermediate  con- 
centrations we  have  to  deal  with  a  rate  of  solution  which  is  com- 
pounded of  the  separate  rates  of  solution  of  the  two  types  of 
molecules.  With  increasing  concentration  of  alcohol  the  rate  of 
solution  of  the  single  molecules  is  progressively  diminished,  but 
that  of  the  double  molecules  is  increased,  owing  to  the  fact  that 
the  proportion  of  double  to  single  molecules  coming  off  from  the 
internal  surfaces  of  the  casein  particles  is  being  increased  suffi- 
ciently rapidly  to  more  than  compensate  for  the  retardation  of 
the  total  rate  of  solution  by  the  alcohol.  At  8  mol.  concentra- 
tion, so  large  a  proportion  of  the  molecules  coming  off  are  of  the 
double  type  that  thereafter  the  retarding  effects  of  increasing 
concentration  of  alcohol  upon  the  total  rate  of  solution  or  pene- 
tration more  than  compensates  for  any  further  increase  in  the 
proportion  of  double  molecules. 

At  intermediate  concentrations  the  quantity  of  casein  dis- 
solved at  first  increases  and  thereafter  diminishes  with  time, 
casein  which  is  initially  dissolved  being  later  reprecipitated. 


290  PHYSICAL  PROPERTIES 

This  can  only  be  interpreted  by  supposing  that  the  condition  of 
the  bulk  of  the  solution  outside  the  casein  particles  differs,  at 
any  rate  for  some  portion  of  the  time  occupied  in  solution,  from 
the  condition  of  that  portion  of  the  solvent  which  has  actually 
penetrated  the  protein  particles.  We  know  that  any  substance 
such  as  alcohol,  which  reduces  the  tension  of  a  solid-water  inter- 
face, tends  to  become  concentrated  at  such  an  interface  (102). 
Hence  at  the  surfaces  of  the  casein  particles  the  concentration 
of  alcohol  will  be  greater  than  in  the  bulk  of  the  fluid.  Suppose 
that  the  concentration  of  alcohol  in  the  bulk  of  the  fluid  be  such 
that  at  equilibrium  the  proportion  of  double  molecules  is  50 
per  cent.  If,  now,  the  concentration  of  alcohol  at  the  surface 
of  the  casein  particles  be  such  as  to  lead  to  the  formation  of  60 
per  cent  of  double  molecules,  then  out  of  every  thirty  molecules 
coming  off  from  the  interior  surfaces  of  the  casein  particles 
eighteen  will  be  of  the  double  type  and  only  twelve  of  the  single 
type.  As  these  come  out  into  the  bulk  of  the  liquid,  equilibrium 
will  tend  to  be  re-established;  resulting  in  the  formation  of  sixteen 
double  and  sixteen  single  molecules,  with  an  increase  in  the  total 
number  of  molecules  to  thirty-two.  This  equilibrium  may  be 
supposed  to  be  slowly  established,  and  meanwhile  the  rate  of 
solution  of  the  casein  has  fallen  very  low;  that  is,  the  fall  in 
concentration  or  concentration  gradient  from  within  the  casein 
particles  to  the  bulk  of  the  fluid  outside  is  very  small.  By  this 
time,  however,  as  a  result  of  depolymerization,  the  molecular 
concentration  of  casein  in  the  outer  fluid  has  actually  become 
greater  than  that  in  the  saturated  solution  which  fills  the  interior 
spaces  of  the  casein  particles.  Thus  the  concentration  gradient 
has  become  negative,  and  as  the  more  concentrated  casein  solu- 
tion diffuses  back  into  the  less  concentrated  but  nevertheless 
saturated  solution  filling  the  interspaces  of  the  sponge-like  par- 
ticles of  casein,  the  excess  of  casein  must  be  reprecipitated.  When 
the  concentration  of  alcohol  is  sufficiently  great,  so  that  the 
molecular  condition  of  the  casern  is  practically  the  same  within 
and  without  the  particles,  a  negative  concentration  gradient  can 
never  arise  and  the  curve  of  solution  reassumes  the  normal  form, 
representing  now,  however,  the  curve  of  solution  of  double  mole- 
cules instead  of  single  molecules  as  at  lower  concentrations  of 
alcohol. 
This  interpretation  of  the  above  facts  finds  further  confirma- 


PASSAGE  INTO  SOLUTION  291 

tion  in  the  effect  of  glycerol  upon  the  rate  of  solution  of  casein 
in  dilute  alkali.  In  glycerol  we  have  a  substance  which,  like 
alcohol,  reduces  the  tension  of  a  solid-water  interface  but  which, 
unlike  alcohol,  does  not  coagulate  (polymerize)  proteins  in  solu- 
tion. Accordingly  we  find  that  glycerol  decreases  the  rate  of 
solution  of  casein  progressively  as  its  concentration  increases. 
No  sign  of  reprecipitation  of  dissolved  casein  is  observed  in  any 
of  the  mixtures.  The  penetration  formula  x  =  Ktm  applies  to 
the  rate  of  solution  in  all  of  the  mixtures  employed  and  the  value 
of  the  coefficient  of  penetration  progressively  decreases  in  a 
smooth  curve  with  decreasing  acceleration  as  the  concentration 
of  glycerol  increases. 

These  results  illustrate  the  part  which  may  be  played  by  capil- 
lary phenomena  in  heterogeneous  systems  which  contain  proteins, 
the  importance  of  which  has,  of  recent  years,  been  especially 
insisted  upon  by,  among  others,  Wo.  Ostwald  and  H.  Freund- 
lich  (67). 

It  is  of  interest  to  estimate,  by  exterpolation  from  the  pene- 
tration formula,  the  amount  of  time  which  would  be  required  to 
"saturate"  an  alkaline  solution  with  casein  by  stirring  up  excess 
of  undissolved  casein  in  it.  We  have  seen  that  the  relation  be- 
tween the  percentage  ( =  x)  of  casein  dissolved  in  a  given  solution 
of  alkali  and  the  time  ( =  t)  of  stirring  is  expressed  by  the  equation 
x  =  Ktm.  For  a  mixture  of  5  grams  of  solid  casein  with  100  cc. 
of  0.00870  N  KOH  the  values  of  K  and  m  at  18-20  degrees  are, 
respectively,  1.48  and  0.146.  The  quantity  of  casein  required 
to  " saturate"  100  cc.  of  this  solution  would  be  7.63  grams. 
Calculating  the  value  of  t  corresponding  to  this  value  of  x  we 
find  that  it  would  take  no  less  than  thirty-one  years  to  fully 
"saturate"  the  solution  with  casein,  at  the  rate  of  stirring  em- 
ployed.* This  very  clearly  indicates  the  great  importance  which 
the  time  factor  may  assume  in  heterogeneous  systems  which 
contain  proteins. 

*  For  more  dilute  solutions  of  alkali  mixed  with  the  same  number  (5)  of 
grams  of  casein  per  100  cc.  the  time  required  for  "saturation"  would  appear 
to  be  shorter,  thus  for  0.00435  N  KOH,  calculating  as  above,  it  is  only  about 
17  days.  This  is  because  not  only  the  concentration  of  alkali  but  also  the 
mass  of  casein,  as  we  have  seen,  plays  a  part  in  determining  the  rate  of  solu- 
tion. The  ratio  of  casein  to  alkali  was  of  course  greater  in  the  more  dilute 
alkaline  solutions  in  inverse  proportion  to  the  concentration  of  the  alkali. 


292  PHYSICAL  PROPERTIES 

2.  The  Swelling  of  Protein  Jellies.  —  A  phenomenon  which  is 
doubtless  very  closely  allied  to  that  of  the  solution  of  solid  pro- 
teins is  the  swelling  or  taking  up  of  water  which  protein  jellies 
undergo  when  immersed  in  water  or  in  certain  watery  solutions. 

When  a  plate  of  water-poor  gelatin  is  immersed  in  water, 
especially  if  the  water  contains  a  little  added  acid  or  alkali,  the 
plate  takes  up  a  very  considerable  quantity  of  water,  many 
times  its  own  weight,  and  at  the  same  time  swells  to  relatively 
enormous  dimensions. 

It  was  first  pointed  out  by  Quincke  (80)  that  the  swelling  of 
gelatin  is  accompanied  by  a  volume  contraction,  that  is,  the 
volume  of  the  swollen  jelly  is  less  than  the  sum  of  the  volumes 
of  the  original  unswollen  jelly  and  the  absorbed  water,  and  it 
was  further  pointed  out  by  Wiedemann  and  Ludeking  (107) 
that  this  process  is  accompanied  by  a  disengagement  of  heat. 
Careful  measurements  of  the  heat  liberated  and  the  volume 
contraction  during  the  swelling  of  casein  in  water  have  been 
made  by  Katz  (46)  who  finds  that  the  relationship  between  the 
heat  liberated  (=  W)  and  the  amount  of  water  (=  i  grams) 
taken  up  by  one  gram  of  protein  is  expressed  by  the  equation: 


in  which  A  and  B  are  constants.     This  implies  that  at  the  initial 
moment  of  the  swelling  process,  the  heat  liberated  by  one  gram 

of  protein  per  gram  of  water  absorbed  will  be  equal  to  the  ratio 

^ 

^,  which  in  this  case  equalled  265  Cal.     The  volume-contraction 

£> 

follows  an  analogous  relationship: 


. 
Jf/M 

in  which  /  and  g  are  constants  and  we  may  similarly  calculate, 
from  the  ratio  -  the  value  of  C0,  or  the  volume  contraction  per 

gram  of  protein  per  gram  of  water  absorbed  at  the  moment  of 
initiation  of  the  process,  and  this  calculation  yields  the  value 
0.3  cc. 

The  time-relations  and  equilibria  in  the  process  of  the  swelling 
of  gelatin  have  been  especially  studied  by  Hofmeister  (40),  Pauli 


SWELLING  293 

(70)  (71)  (72),  Wo.  Ostwald  (64)  (65),  Chiari  (16),  Ehrenberg 
(23),  Procter  (74)  (75)  (76)  (77)  (78),  and  Lenk  (50). 

Hofmeister  found  that  the  swelling  of  gelatin  plates  proceeds, 
at  first  rapidly  and  then  more  slowly,  until  it  attains  a  maximum 
which  is  a  function  of  the  thickness  and  weight  of  the  plate. 
After  the  attainment  of  this  maximum  the  superficial  layers  of 
the  plate  tend  to  go  into  solution,  especially  if  the  water  be  acid 
or  alkaline,  and  the  plate,  in  consequence  loses  weight.  Desig- 
nating by  the  symbol  W  the  weight  of  water  which  unit  weight 
of  the  gelatin  absorbs  from  water  or  a  watery  solution  in  t 
minutes,  Hofmeister  found  that  the  empirical  equation: 


W-P   1-— l—  \  (v) 


applies  with  tolerable  accuracy,  P  being  the  maximum  amount 
of  water  which  unit  weight  of  the  gelatin  plate  will  imbibe  (the 
swelling-maximum),  c  a  constant  and  d  the  thickness  in  milli- 
meters of  the  plate  at  its  maximal  degree  of  swelling.  This 
leads  to  the  conclusion  that  the  initial  velocity  of  swelling  is 
proportional  to  the  amount  of  swelling  which  the  plate  is  able 
to  undergo  and  that  it  therefore  decreases  regularly  as  the  degree 
of  swelling  approaches  more  and  more  nearly  the  maximum. 

Pauli  assumed  that  each  particle  of  the  gelatin  takes  up  water 
from  every  neighboring,  more  fully  swollen  particle  at  a  velocity 
proportional  to  the  difference  between  their  water  contents.  This 
leads  to  the  equation: 

v         1     i     M  -  Q 


where  Q  is  the  quantity  of  water  taken  up  by  unit  weight  of 
gelatin  in  time  t,  M  is  the  maximum  degree  of  swelling  which 
the  gelatin  attains,  Qi  is  the  quantity  of  water  taken  up  by  unit 
weight  of  gelatin  in  time  ti  and  K  is  a  constant  which  varies 
inversely  with  the  thickness  of  the  plate. 

Hofmeister's  formula  leads  to  the  conclusion  that  the  velocity 
of  swelling  is  at  every  instant  proportional  to  the  square  of  the 
swelling  which  the  plate  has  yet  to  undergo,  Pauli's  to  the  con- 
clusion that  the  velocity  of  swelling  is  at  every  instant  propor- 
tional to  the  first  power  of  the  same  quantity  (termed  by  Pauli 
the  "  swelling  deficit")-  Both  formulae  lead  to  the  conclusion 


294  PHYSICAL  PROPERTIES 

that  the  velocity  of  swelling  is  at  a  maximum  at  the  instant  of 
immersion  and  therefore  decreases  with  time.  This  corresponds 
to  the  experimental  facts. 

As  we  shall  see  in  the  next  chapter,  the  proteins  exert  a  small, 
but  definite,  osmotic  pressure.  They  are  at  the  same  time  not 
diffusible  through  colloids,  or  only  very  slightly  so.  Any  crystal- 
loids which  may  be  present  in  the  external  fluid  which  bathes 
the  gelatin  can  penetrate  the  gelatin  albeit,  possibly,  more  slowly 
than  the  water.  The  gelatin  plate,  therefore,  acts  like  an  osmom- 
eter  which  provides  its  own  membrane  which  is  permeable  to 
crystalloids  and  not  to  colloids.  Hence  osmotic  forces  must 
play  a  part  in  the  taking  up  of  water  by  protein  jellies.  A  phe- 
nomenon in  the  domain  of  crystalloids  which  presents  some 
analogies  to  this  aspect  of  the  swelling  of  colloids  is  the  following:* 
If  we  place  at  the  bottom  .of  a  column  of  distilled  water  a  layer 
of  phenol  and  introduce  below  this  a  layer  of  a  saturated  solution 
of  KC1  in  water  and  now  allow  the  system  to  stand  at  constant 
temperature  the  layer  of  phenol  gradually  moves  up  the  column 
of  water;  in  other  words  the  layer  of  solution  below  the  phenol 
"swells."  The  solvent,  water,  being  soluble  in  phenol,  the  phenol 
is  permeable  to  it,  while  the  KC1  being  insoluble  in  phenol,  cannot 
pass  through  the  layer  of  phenol. 

Not  only  osmotic,  but  also  chemical  phenomena  must,  however, 
play  a  part  in  the  swelling  of  protein  jellies.  As  we  have  seen  in 
Chaps.  VI  and  XI  the  passage  of  a  protein  into  solution  involves 
the  addition  of  the  elements  of  water  to  terminal  —  NH2  and 
—  COOH  groups  and  also,  possibly,  to  internal  —  N.HOC  — 
groups,  resulting  in  the  depolymerization  of  the  protein.  Not 
only  osmotic  phenomena  but  hydration  of  the  gelatin  must  there- 
fore occur  in  the  process  of  swelling.  In  evidence  of  the  correctness 
of  this  view  Pauli  points  to  the  fact  that,  according  to  Weidemann 
and  Liideking  (loc.  cit.),  the  swelling  of  gelatin  is  accompanied 
by  a  disengagement  of  heat,  while  the  solution  of  gelatin  is  ac- 
companied by  an  absorption  of  heat.  Evidently  the  processes  of 
solution  and  swelling  are  each  composed  of  two  factors,  one 
leading  to  a  disengagement  and  the  other  to  the  absorption  of 
heat.  The  former  process  is,  Pauli  believes,  the  chemical  binding 
of  water  by  the  protein,  the  latter  the  passage  of  the  hydrated 

*  To  which  my  attention  was  drawn,  in  this  connection,  by  my  colleague 
Dr.  F.  G.  Cottrell. 


SWELLING  295 

protein  into  solution  (or,  in  swelling,  the  osmotic  inhibition  of 
water).  In  swelling  the  chemical  heat-effect  predominates;  in 
the  dissolving  of  the  gelatin,  the  heat-effect  of  solution. 

That  a  part,  at  least,  of  the  water  in  swollen  gelatin  is  chemi- 
cally bound  by  the  protein  is  shown  by  the  following  experiment 
(74).  If  two  gelatin  plates  be  brought  to  the  same  degree  of 
swelling,  the  one  in  neutral  and  the  other  in  acid  water,  and  if 
they  be  then  immersed  in  absolute  alcohol,  the  alcohol  will  ab- 
stract all  of  the  water  from  the  plate  which  has  been  swollen  in 
neutral  water,  but  not  from  the  plate  which  has  been  swollen 
in  acid  water.  Now,  as  we  have  seen  in  Chap.  V,  the  fact  that 
a  constituent  of  a  system  can  be  completely  removed  from  it  by 
washing  out  with  an  appropriate  solvent  affords  no  valid  proof 
that  the  constituent  in  question  did  not  exist,  within  the  system, 
in  a  state  of  chemical  combination.  But  if,  on  the  contrary,  it 
should  prove  impossible  or  exceptionally  difficult  to  remove  the 
constituent  by  washing  with  a  fluid  in  which  it  is  very  soluble, 
then  the  prima  facie  evidence  that  it  exists  in  the  system  in  a 
condition  of  chemical  union  is  very  strong.  We  may  conclude, 
therefore,  that  when  gelatin  is  swollen  in  acid  water  a  part  of 
the  water  which  is  taken  up  by  the  gelatin  is  chemically  bound 
by  it;  and  we  have  no  valid  reason  for  supposing  that  the  water 
taken  up  from  neutral  solutions  is  not  similarly  bound  although 
less  firmly. 

The  equilibria  attained  in.  the  swelling  of  gelatin  in  solutions 
of  acids  have  recently  been  very  thoroughly  investigated  by 
Procter.  This  investigator  has  found  that  gelatin  absorbs  both 
acid  and  water  from  acid  solutions,  but  absorbs  the  acid  in  excess, 
so  that  the  proportion  of  acid  in  the  surrounding  fluid  diminishes. 
If  the  initial  concentration  of  acid  in  the  external  fluid  lies  be- 
tween 0.01  and  0.25  N,  then  assuming  that  at  the  end  of  the  process 
(attainment  of  maximal  swelling)  the  concentration  of  free  acid 
is  the  same  within  and  without  the  jelly,  the  amount  of  acid 
which  is  "bound"  by  the  gelatin  is  0.7  to  0.8  X  10~3  (=  70  to 
80  X  10~5)  equivalents  per  gram.  The  equivalence,  at  the 
attainment  of  maximal  swelling,  is  the  same  for  all  strong  acids 
but  falls  below  this  value  for  weak  acids.  While  the  proportion 
of  acid  which  is  "bound"  by  the  gelatin  varies  but  slightly  with 
the  concentration  of  the  acid  in  the  surrounding  fluid,  this  is  not 
true  of  the  degree  of  swelling  attained,  which  in  strongly  acid 


296  PHYSICAL  PROPERTIES 

solutions  attains  its  maximum  at  a  dilution  below  that  required 
for  complete  fixation  of  the  acid  by  the  gelatin,  and  then  falls  in 
a  continuous  curve  with  increasing  concentration  of  the  external 
acid  solution.  Procter  interprets  this  by  supposing  that  the 
swelling  of  the  gelatin  is  determined  by  two  opposing  forces,  the 
osmotic  pressure  of  the  acid-gelatin  compound  imprisoned  by 
its  indiffusibility  within  the  jelly  being  balanced  by  the  osmotic 
pressure  of  the  acid  outside.  That  the  acid  in  the  outer  fluid 
does  exert  an  osmotic  pressure  at  the  surface  of  the  jelly  Procter 
infers  from  the  fact  that  concentrated  neutral  salts  contract  the 
jelly  to  a  horny  consistency  and  expel  the  associated  (imbibed 
or  combined)  water  without  affecting  the  acid-gelatin  compound. 
In  view  of  the  considerations  set  forth  in  Chap.  VI  we  can  see, 
however,  that  the  assumption  that  the  external  acid  exerts  an 
osmotic  pressure  is  an  unnecessary  one  and,  indeed,  inconsistent 
with  the  fact  that  the  acid  freely  penetrates  the  gelatin  and 
combines  with  it.  In  view  of  the  known  dependence  of  coagula- 
tion upon  phenomena  of  dehydration  (Cf.  Chap.  VI)  and  the  fact 
that  protein  salts  may  be  coagulated  as  such  and  without  decom- 
position, we  may  infer  that  the  shrinkage  of  gelatin  jellies  upon 
addition  of  concentrated  salts  to  the  external  medium  is  not 
due  to  the  osmotic  pressure  of  the  salts,  since  gelatin  is  known 
to  be  permeable  to  inorganic  ions,  but  to  the  competition  between 
the  inorganic  salt  and  the  gelatin  salt  for  water. 

Procter  has  taken  advantage  of  the  fact  that  the  acid-gelatin 
compound  is  coagulated  without  decomposition  by  concentrated 
salt  solutions  to  determine  the  quantity  of  acid  " bound"  by 
gelatin  in  acid  solutions  of  varying  concentration.  He  finds 
that  the  proportion  of  acid  which  is  bound  per  unit  mass  of 
gelatin  does  not  agree  with  the  requirements  of  the  Ostwald 
dilution-law  for  a  binary  electrolyte,  no  values  of  the  hydrolytic 
dissociation-constant  and  the  molecular  weight  yielding  calcu- 
lated results  which  are  consistent  with  the  initial  rapid  and 
subsequent  slow  rise  in  combining  capacity  with  increasing  con- 
centration of  acid  in  the  surrounding  medium.  Better  agree- 
ment between  theory  and  experiment  is  attained  by  assuming 
that  gelatin  behaves  as  a  diacid  base  having  a  molecular  weight 
of  839,  which  agrees  with  the  molecular  weight  estimated  by 
Paal  from  freezing-  and  boiling-point  measurements  (68). 

The  taking  up  of  water  by  gelatin  from  acid  solutions  is  ac- 


SWELLING  297 

counted  for  by  Procter  in  much  the  same  manner  as  that  out- 
lined above  (76).  He  pictures  the  gelatin  acid-compound  as  a 
coherent  mass  from  which  the  gelatin  molecules  cannot  diffuse  or 
separate  and  which  in  most  respects  behaves  like  a  single  enor- 
mous complex  molecule.  It  is  reasonable,  he  considers,  to  visual- 
ize it  as  a  felted  mass  of  amino-acid  chains  held  to  each  other  by 
attractions  which  possibly  attach  only  their  ends,  but  freely 
admitting  the  passage  of  liquid  between  them.  He  assumes,  in 
accordance  with  our  former  conceptions  of  the  mode  of  forma- 
tion and  ionization  of  protein  salts,  that  the  compound  yields 
acid  anions,  but  these  anions,  although  diffusible,  are  held  within 
the  mass  by  electrostatic  forces,  since  they  cannot  pass  beyond 
the  sphere  of  attraction  of  their  companion  colloid  ions  which 
form  the  jelly  and  which  are  therefore  immobilized.  The  only 
way,  therefore,  in  which  the  osmotic  pressure  of  the  anions  can 
take  effect  is,  not  by  their  movement  but  by  the  movement  of 
water,  resulting  in  the  swelling  of  the  entire  jelly  mass  and  its 
dilution  by  admixture  with  the  outside  solution. 

Two  very  serious  objections  attach  to  this  interpretation  of 
the  phenomena.  In  the  first  place,  as  Procter  himself  has  pointed 
out,  were  this  the  actual  mechanism  of  swelling,  then  the  opera- 
tive force  compelling  movement  of  the  water  would,  in  ultimate 
analysis,  be  the  electrostatic  tension  which  prevents  the  acid 
anions  from  moving  outwards  into  the  surrounding  solvent. 
There  should  thus  be  a  measurable  potential  difference  between 
the  gelatin  jelly  and  the  external  medium.  This  potential  differ- 
ence has  been  sought  for  by  Ehrenberg  who  was  unable  to  detect 
any  measurable  potential  between  the  interior  of  a  jelly  and  the 
external  medium  (23).  In  the  second  place,  as  Procter  also 
points  out,  another  difficulty  lies  in  the  fact  that  the  condition 
which  would  thus  arise  would  offer  no  equilibrium,  since  the 
acid  anions  and  the  free  acid  itself  could  not  simultaneously 
be  equal  in  concentration  within  and  without  the  jelly.  Our 
more  recent  views  regarding  the  mode  of  formation  and  ionization 
of  protein  salts  reconcile  both  these  difficulties,  however,  for, 
since  we  may  assume  that  no  inorganic  ions,  or  at  most  a  very 
small  proportion,  are  yielded  by  the  protein-acid  compound,  the 
swelling  of  the  jelly  must  be  due,  just  as  it  is  in  the  case  of  gel- 
atin immersed  in  neutral  water,  to  the  osmotic  pressure  of  the 
colloid  particles  themselves,  which,  being  unable  to  penetrate 


298  PHYSICAL  PROPERTIES 

the  colloid  network  in  which  they  are  entangled  necessarily 
compel  the  compensating  migration  of  water.  No  electro- 
static tension  between  the  jelly  and  the  external  solution  need 
be  assumed  and  since  no  acid  anions  are  yielded  by  the  protein 
salts,  simultaneous  equality  of  concentration  of  the  uncombined 
acid  and  the  acid  anions  within  and  without  the  jelly  will  be 
assured  by  their  normal  and  equal  diffusion  into  the  jelly.  The 
increased  swelling  capacity  of  gelatin  in  solutions  of  acids  or 
alkalies  is  merely  the  expression  of  the  fact  that  the  ionization 
of  the  protein  salt  leads  to  an  increase  in  the  number  of  colloid 
particles  per  unit  volume  of  the  jelly  and  possibly  also  in  part 
of  the  fact  that  protein  ions  have  a  greater  affinity  for  water 
than  undissociated  protein  molecules. 

This  conception  of  the  process  of  swelling  would  still  yield 
no  equilibrium  or  swelling-maximum  were  there  no  compensating 
force  acting  in  an  opposite  sense  to  the  osmotic  pressure  of  the 
gelatin.  Since  gelatin  plates  when  immersed  in  water  do  not 
swell  indefinitely  until  swelling  merges  insensibly  into  solution 
but,  on  the  contrary,  display  a  more  or  less  well-marked  swelling- 
maximum,  the  osmotic  pressure  exerted  by  the  colloid  particles 
within  the  jelly  must,  upon  attainment  of  this  maximum,  be 
balanced  by  an  equal  opposing  force  which  Procter  interprets 
as  the  tension  of  the  elastic  colloid  network  (77).  Applying 
Hooke's  law,'  Procter  finds  that  this  tension  (=  E)  is  defined 
by  the  equation : 


TjJ    C1  I   V - 

\         sp.  gr.  of  gelatin/ ' 

where  E  is  the  stress  inducing  swelling,  V  is  the  volume  attained 
by  one  gram  of  gelatin,  and  C  is  the  modulus  of  elasticity.  He 
finds  that  C  =  0.00125  at  7  degrees;  0.00055  at  15  degrees;  and 
0.00021  at  18  degrees.  The  value  of  E  at  first  increases  with 
acidity  to  a  maximum  and  then  decreases,  slowly  approaching 
zero. 

It  has  been  shown  by  Ostwald  (64)  and  Chiari  (16)  that  gelatin 
in  very  faintly  acid  media  displays  a  well-marked  minimum  of 
swelling-capacity.  This  minimum  occurs  at  a  hydrogen  ion  con- 
centration almost  exactly  coinciding  with  the  hydrogen  ion 
concentration  of  gelatin  solutions  in  which  the  gelatin  is  "iso- 
eleetric,"  i.e.,  does  not  wander  in  an  electrical  field.  In  other 


GELATINIZATION  AND  COAGULATION  299 

words  minimal  combining  capacity  and  ionization  of  the  jelly 
is  accompanied  by  minimal  swelling  capacity. 

Hofmeister  has  studied  the  effects  of  salts,  acids  and  bases 
upon  the  rate  and  magnitude  of  the  swelling  which  gelatin  plates 
undergo  when  immersed  in  their  solutions;  we  have  had  occasion 
to  comment  upon  the  significance  and  interpretation  of  his  results 
in  Chap.  VI.  Wo.  Ostwald  (65)  has  investigated  the  influence 
of  the  concentration  of  added  salts  upon  the  swelling  of  gelatin 
plates  in  water.  He  asserts  that  the  degree  of  swelling  attained 
after  a  given  period  does  not  vary  continuously  in  a  definite 
sense  as  the  concentration  of  the  salt  is  increased,  the  curve 
displaying  the  influence  of  the  concentrations  of  the  salt  upon  the 
degree  of  swelling  exhibiting  very  marked  maxima  and  minima. 
We  shall  have  occasion  to  further  dwell  upon  the  significance 
of  these  results  in  the  next  chapter. 

The  phenomena  which  accompany  and  the  processes  which 
underlie  the  taking  up  or  loss  of  water  by  living  tissues  have  been 
investigated  by  Loeb  (52)  (53),  Cooke  (21),  Bottazzi  and  Scalinci 
(6),  Beutner  (3),  and  Korosy  (47).  Bottazzi  and  Scalinci  have 
drawn  attention  to  the  remarkable  fact,  previously  pointed  out 
by  von  Schroeder  in  the  case  of  gelatin  (93),  that  the  crystalline 
lens  swells  very  markedly  in  distilled  water  or  physiological 
saline  solution,  but  progressively  loses  water  when  suspended  in 
saturated  water  vapor  at  the  same  temperature.  This  phe- 
nomenon undoubtedly  involves  a  departure  from  the  second  law 
of  thermodynamics,  realizing  the  possibility  which  was  depicted 
figuratively  by  "Maxwell's  demon."  It  is  not  surprising,  perhaps, 
that  the  exception  to  the  rule  should  have  been  first  encountered 
in  the  domain  of  colloid  jellies  in  which  definite  structures  of 
molecular  dimensions  play  so  pronounced  a  r61e  in  determining 
their  behavior. 

The  significance  of  the  phenomena  of  swelling  in  certain  aspects 
of  clinical  medicine  has  been  especially  insisted  upon  by  M.  H. 
Fischer  and  discussed  by  him  at  length  in  his  work  upon  cedema 
(26).  Exception  has,  however,  been  taken  by  Moore  to  certain  as- 
pects of  Fischer's  interpretation  of  the  phenomena  of  cedema  (58). 

3.  The  Gelatinization  and  Coagulation  of  Proteins.  —  If  an 
insoluble  gel,  such  as  white  of  egg  coagulated  by  fixatives,  the 
gel  of  collodion  produced  by  the  action  of  chloroform  upon  an 
ether  solution,  common  black  india-rubber,  or  the  hydrogel  of 


300  PHYSICAL  PROPERTIES 

silica,  be  examined  under  high  magnification  they  can  all  be 
demonstrated,  Hardy  states  (33)  (34),  to  possess  a  fine  open 
spongy  structure.  When,  for  example,  a  13  per  cent  solution 
of  egg-white  is  fixed  with  sublimate,  sections  are  found  to  show 
a  sponge  or  net  structure.  Staining  the  section  with  iron- 
hsematoxylin,  with  saturated  solutions  of  acid  and  basic  dyes  or 
even  by  evaporation  to  dryness  in  solutions  of  such  dyes,  failed 
to  produce  the  staining  of  any  substance  within  the  meshes  of 
the  net,  while  pressure  applied  to  the  gel  resulted  in  the  squeezing 
of  fluid  out  of  these  interstices.  The  structure  of  the  gel  is 
therefore  that  of  an  open  sponge-work  of  solid,  containing  fluid 
within  its  meshes.  Direct  experimentation  with  agar  showed 
that  in  a  gel  containing  1  per  cent  agar  the  solid  framework  is 
a  solution  of  water  in  agar,  while  the  fluid  contained  in  the  inter- 
stices is  a  dilute  solution  of  agar  in  water;  upon  the  heating  the 
system  the  two  components  become  miscible  in  each  other  and 
we  obtain  a  homogeneous  solution.  Upon  the  basis  of  these 
facts  Hardy  draws  a  far-reaching  analogy  between  this  system 
(and  other  jellies  which  are  heat-reversible)  and  the  system 
phenol-water,  which,  if  it  contains  more  than  71  per  cent  or  less 
than  76  per  cent  of  phenol  separates,  at  temperatures  below 
80  degrees,  into  two  phases,  the  one  a  solution  of  phenol  in  water, 
the  other  a  solution  of  water  in  phenol.  According  to  the  view 
developed  by  Hardy,  the  two  cases  differ  only  in  the  fact  that 
upon  separation  of  the  two  phases  in  the  agar-water  system  the 
system  retains  a  structure,  while  in  the  phenol-water  system  no 
structure  is  retained.  Essentially,  he  believes,  the  difference 
between  the  two  systems  consists  in  this,  that  when  the  phenol- 
water  system  separates  into  two  phases  the  phases  become  sepa- 
rated by  the  minimum  possible  surface,  namely  a  plane;  while 
when  the  agar-water  system  separates  into  two  phases  they 
remain  in  contact  over  an  area  far  larger  than  the  minimum.  In 
the  latter  case  it  would  appear  that  the  surface  tension  at  the 
surface  of  separation  of  the  two  phases  is  very  low,  so  that  the 
force  leading  to  a  diminution  of  surface  is  indefinitely  small. 

Pauli  and  Rona  (72)  object  to  the  use  which  Hardy  has  made 
of  the  term  "phase"  in.  this  connection.  They  point  out  that 
the  fluid  which  may  be  pressed  out  of  an  agar  jelly  may  contain 
from  0  to  0.14  per  cent  of  agar  according  to  the  magnitude  and 
mode  of  application  of  the  pressure,  and  they  urge,  in  considera- 


GELATINIZATION  AND  COAGULATION  301 

tion  of  this  fact,  that  there  can  be  no  sharp  separation  into  phases 
within  an  agar,  and,  presumably,  within  a  gelatin  jelly.  They 
believe  that  a  sharp  distinction  should  be  drawn  between  the 
coagulation  of  a  protein  by  dehydrating  agents  and  the  gelatin- 
ization  of  its  solution,  since  in  the  former  case  a  sharp  separation 
into  two  phases  occurs  while  in  the  latter  it  does  not.  The 
experimental  basis  of  this  objection  to  Hardy's  view  is,  however, 
not  altogether  a  sufficient  one,  since  if  any  two  parts  of  a  chemical 
system,  which  are  of  more  than  molecular  thickness,  are  sepa- 
rated by  a  surface,  they  constitute,  in  the  sense  of  the  phase-rule, 
separate  phases.  The  observation  of  Pauli  and  Rona  shows, 
however,  that,  at  least  in  agar  jellies,  if  separate  phases  exist 
they  are  not  of  definite  or  constant  composition. 

The  fact  that  in  protein  jellies  which  do  consist  of  two  phases 
the  phases  are  not  of  constant  composition  is  also  very  clearly 
revealed  by  Hardy's  own  results,  of  which  a  description  follows: 

The  manner  in  which  the  structure  of  an  insoluble  gel  is  built 
up  can,  according  to  Hardy,  be  readily  observed  in  the  ternary 
mixture  alcohol,  gelatin,  and  water.  If  13.5  grams  of  gelatin 
are  mixed  with  50  cc.  of  water  and  50  cc.  of  absolute  alcohol, 
a  mixture  is  formed  which  is  optically  homogeneous  at  17  to  20 
degrees  but  which  separates  into  two  phases  at  temperatures 
below  this.  "As  the  temperature  falls  below  the  limit  a  clouding 
occurs  which  I  find  to  be  due  to  the  appearance  of  fluid  droplets 
which  gradually  increase  in  size  until  they  measure  3  ju.  On 
cooling  further,  these  fluid  droplets  become  solid  and  they  begin 
to  adhere  to  one  another.*  The  framework  is  therefore  an  open 
structure  which  holds  the  fluid  phase  in  its  interstices."  "  When 
once  formed  the  phases  have  considerable  stability.  If  the  drop- 
lets are  composed  of  a  solid  solution  one  may,  by  the  addition 
of  water,  cause  them  to  increase  to  relatively  vast  dimensions 
without  their  being  destroyed;  as  they  increase  in  size  their 
refractive  index  approximates  more  and  more  to  that  of  the 
external  phase  until  t  ley  are  finally  lost  sight  of.  The  addition 
of  alcohol,  however,  once  more  brings  them  into  view  and  causes 
them  to  shrink.  Owing  to  this  stability,  once  a  configuration 
has  been  established,  one  has  to  far  overstep  the  conditions  of 
its  formation  in  order  to  destroy  it.  This  would  account  for 

*  The  formation  of  similar  droplets  has  been  observed  by  Pauli  and  Rona 
(72)  in  the  coagulation  of  gelatin  by  salts. 


302  PHYSICAL  PROPERTIES 

the  remarkable  hysteresis  observed  in  reversible  gels.  .  .  .  When 
water  is  added  to  a  ternary  mixture  so  as  to  considerably  swell 
the  droplets  the  system  is  unstable  and  the  two  phases  mix  at  once 
when  it  is  mechanically  agitated"  (34). 

In  gels  of  this  type  which  are  dilute  with  respect  to  the  colloid, 
therefore,  the  structure  is  that  of  an  open  sponge- work;  the 
meshes  being  filled  with  water  or  a  water-rich  solution  of  the  sub- 
stance forming  the  gel,  while  the  frame- work  of  the  sponge  con- 
sists of  anastomosing  threads  composed  of  linearly  arranged 
globules  of  the  water-poor  phase.  In  such  gels,  therefore,  the  sur- 
face of  the  water-poor  phase  is  convex  while  that  of  the  water-rich 
phase  is  concave;  in  other  words,  the  water-poor  phase  is  internal 
to  the  water-rich  or  external  phase.  If,  however,  to  a  ternary 
mixture  of  gelatin,  alcohol  and  water  which  forms  such  a  gel 
as  that  described  above,  more  gelatin  be  added,  the  character 
of  the  gel  changes  entirely,  the  water-poor  phase  becomes  con- 
cave and  the  water-rich  phase  instead  of  being,  as  formerly, 
concave  becomes  convex  to  it.  On  cooling  such  a  mixture  to  a 
temperature  below  that  at  which  it  forms  an  optically  homo- 
geneous solution,  droplets  separate  out  which  are  poor  in  gelatin, 
while  the  interstitial  portion  of  the  system,  which  is  rich  in 
gelatin,  solidifies.  Thus  the  gel  comes  to  possess  a  honeycomb 
structure,  the  droplets  being  poor  in  gelatin,  rich  in  water.  This 
is  very  clearly  shown  in  the  following  determinations  of  Hardy. 

TEMPERATURE  OF  THE  MIXTURE  15  DEGREES 

(Equal  parts  of  water  and  alcohol) 


Per  cent  gelatin  in 
mixture 

Per  cent  gelatin  in 
droplets  (internal) 
phase) 

Per  cent  gelatin  in 
interstices  (external 
phase) 

6.7 
13.5 
36.5 

17.0 
18.0 
8.5 

2.0 
5.5 
40.0 

From  these  determinations  it  is  also  clear,  as  I  have  said, 
that  the  two  phases  are -not  of  constant  composition,  but  may, 
under  different  conditions  of  total  concentration,  etc.,  vary  widely 
in  their  relative  and  absolute  gelatin  and  water  content.  This 
system  differs,  therefore,  from  the  system  phenol-water  not  only 


GELATINIZATION  AND  COAGULATION  303 

in  the  extent  of  the  surface  which  separates  the  phases,  but  also 
in  the  variability  of  the  composition  of  its  phases,  in  this  respect 
resembling  rather  the  system  hydrated  silica-water,  investigated 
by  van  Bemmelen  (1). 

An  inversion  of  the  external  and  internal  phases  of  a  diphasic 
system,  similar  to  that  observed  by  Hardy  on  increasing  the  con- 
centration of  gelatin  in  the  system  gelatin-alcohol-water,  may  also 
be  observed  in  the  system  olive  oil-alkaline  water,  on  increasing 
the  proportion  of  oil  to  water  (84)  (85),  and  is  here  manifestly 
due  to  the  inability  of  a  limited  quantity  of  alkaline  water  to 
surround  and  envelop  an  unlimited  quantity  of  oil. 

The  question  has  been  raised  whether  the  jelly  which  is  formed 
by  gelatin  dissolved  in  water  (instead  of  alcohol-water  mixtures) 
really  possesses  a  structure  analogous  to  that  observed  by  Hardy 
in  ternary  systems.  It  has  been  urged  that  this  structure  is 
an  artefact  arising  out  of  partial  coagulation  of  the  protein, 
since  it  is  not  directly  visible  in  binary  systems.  The  action  of 
coagulants  upon  jellies  which  already  possess  a  structure  of  this 
type,  however,  is  not  to  otherwise  alter  but  merely  to  coarsen 
the  structure.  This  is  due  to  loss  of  water  on  the  part  of  the 
colloid-rich  droplets  with  a  consequent  diminution  of  the  volume 
of  the  colloid-rich  phase  and  an  increase  in  the  volume  of  the 
more  fluid  interstices.  This  can  be  shown,  not  only  by  direct 
observation,  but  also  by  the  relative  ease  with  which  water  can 
be  expressed  from  the  jelly  before  and  after  " fixation."  From 
Poiseuilles'  law  for  the  outflow  of  liquids  from  capillary  tubes, 
it  follows  that  the  pressure  required  to  express  the  fluid  from  the 
interstices  of  a  gel  at  a  given  rate  must  vary  approximately  as 
the  inverse  fourth  power  of  the  diameter  of  the  meshes,  although, 
of  course,  the  variable  viscosity  of  the  expressed  fluid  will  be  a 
factor  introducing  departures  from  this  simple  law.  Now  a 
hydrogel  containing  13  per  cent  of  pure  gelatin  at  a  tempera- 
ture of  15  degrees  will  endure  a  pressure  of  400  pounds  to  the 
square  inch  without  expression  of  water;  after  fixation  with 
formalin  or  corrosive  sublimate,  however,  the  fluid  can  be  ex- 
pressed from  the  gel  like  water  from  a  sponge,  with  simple  hand- 
pressure  (33). 

Since  more  complete  coagulation  does  not  alter  the  type  of 
structure  possessed  by  jellies  of  partially  coagulated  protein, 
but  merely  coarsens  it,  it  is  a  fair  inference  that  jellies  which 


304  PHYSICAL  PROPERTIES 

have  undergone  no  measure  of  coagulation  also  possess  the  type 
of  structure  outlined  by  Hardy,  but  that  owing  to  its  fineness  the 
details  of  this  structure  are  not  visible. 

The  existence  of  a  structure  in  jellies  formed  by  the  solution 
of  gelatin  in  water  is  also  objectively  demonstrated  by  the  ob- 
servation of  Liesegang  (51)  that  when  silver  nitrate  diffuses 
into  gelatin  which  is  impregnated  with  potassium  bichromate, 
the  precipitation  of  insoluble  silver  bichromate  does  not  occur 
indifferently  in  all  parts  of  the  area  of  diffusion  but  in  concentric 
circles.  It  has  also  been  shown  by  Rohonyi  (91)  that  when 
thin  films  of  gelatin  are  frozen  the  ice  crystals  are  formed  in 
concentric  rings.  It  is  difficult  to  clearly  conceive  any  mecha- 
nism which  would  permit  this  in  a  perfectly  homogeneous  medium. 
The  theory  that  crystallization  is  inhibited  by  the  gelatin  until 
a  certain  degree  of  super-saturation  is  attained  might  account 
for  failure  of  precipitation  or  crystallization  at  certain  points, 
but,  provided  the  jelly  is  strictly  homogeneous  and  structureless 
it  fails  to  account  for  its  appearance  at  other  points.  That  the 
distinction  between  gelation  and  coagulation  is  merely  a  dis- 
tinction of  degree  and  not  of  kind  has  been  shown  by  Buglia  (12). 

The  experiments  of  Hardy  show  that  on  adding  water  to  the 
system  alcohol-water-gelatin,  the  gelatin-rich  phase  progressively 
imbibes  water  until  it  passes  by  a  series  of  insensible  transitions 
into  a  solution  of  gelatin.  Having  regard  to  this  fact  and  to  the 
probability,  which  will  be  indicated  in  the  following  chapter,  that 
the  structure  of  a  jelly  of  uncoagulated  protein  is  merely  the  con- 
tinuation of  a  structure  which  pre-exists  in  the  solutions  which 
become  gelatinized,  it  appears  highly  probable  that  in  uncoagulated 
protein  jellies  the  structure  is  of  molecular  dimensions,  so  that  the 
constituents  of  the  jelly  are  not  separated  from  one  another  by  any 
definite  interface.  This  conception  does  not  in  the  least  militate 
against  the  view  that  the  structure  of  a  coagulated  or  partially 
coagulated  jelly  merely  results  from  the  coarsening  of  a  pre-exist- 
ing molecular  structure.  It  is  probable  that  the  coagulation  of  a 
protein  solution,  without  passing  through  the  intermediate  stage 
of  gelatinization,  is  similarly  accompanied  and  possibly  (from 
a  mechanical  standpoint)  accomplished  by  the  coarsening  of  a 
pre-existing  molecular  structure. 

4.  The  Coagulation  of  Proteins  by  Heat,  Light  and  Hydro- 
static Pressure.  —  Several  aspects  of  the  phenomenon  of  heat- 


HEAT,  LIGHT  AND  PRESSURE  305 

coagulation  have  been  discussed  at  some  length  in  Chap.  VI,  and 
it  has  been  pointed  out  in  that  chapter,  and  incidentally,  in 
Section  1  of  this  chapter  that  the  application  of  heat  to  proteins 
in  solution  results,  probably,  not  only  in  the  abstraction  of  the 
elements  of  water  from  the  protein,  with  the  formation  of  anhyd- 
rides, but  also  in  its  polymerization. 

Under  definite  conditions  of  concentration,  reaction  and  salt- 
content  of  its  solution,  etc.,  the  coagulation-temperature  of  *  a 
protein  is  tolerably  constant,  and  Fredericque  (27),  Halliburton 
(29)  and  others  have  utilized  this,  property  very  extensively  in 
the  endeavor  to  separate  and  characterize  different  proteins. 

The  concentration  of  protein,  and  especially  the  presence  of 
other  substances  in  the  solution,  very  markedly  modifies  the 
coagulation-temperature,  however.  The  influence  of  inorganic 
salts,  upon  the  "temperature  of  coagulation"  has  been  inci- 
dentally discussed  in  Chap.  VI.  The  influence  of  a  variety 
of  salts  in  acid  and  alkaline  solutions  upon  the  coagulation- 
temperature  of  proteins  has  been  very  extensively  studied  by 
Pauli  and  his  pupils  (32),  and  found  to  be  in  satisfactory  accord- 
ance with  the  view  that  heat-coagulation,  like  salt-  or  alcohol- 
coagulation  is  accomplished  through  the  dehydration  of  the 
protein  molecules. 

The  very  important  observation  has  been  made  by  Chick 
and  Martin  (17)  that  the  heat-coagulation  of  haemoglobin  and 
egg  albumin  (in  solutions  of  crystallized  preparations  of  these 
substances)  is  not  an  instantaneous  process,  but  that  it  proceeds 
with  a  definite  velocity  which  decreases  as  the  protein  becomes 
coagulated  and  increases  very  markedly  with  rise  in  temperature. 
The  relation  between  time  of  exposure  to  a  temperature  suffi- 
cient to  bring  about  coagulation  and  the  quantity  of  protein 
coagulated  is,  for  haemoglobin,  that  which  is  characteristic  for 
the  occurrence  of  a  monomolecular  chemical  reaction,  namely: 

log  ^  =  Ktt 

Lt 

where  C0  is  the  initial  concentration  of  the  substance  (protein), 
Ct  its  concentration  at  time  t  and  K  is  a  constant  (the  velocity- 
constant  of  the  reaction).  The  following  are  illustrative  of  their 
results : 


306 


PHYSICAL  PROPERTIES 


COAGULATION  OF  HAEMOGLOBIN,  3  PER  CENT  SOLUTION 


Experi- 

Temp, 

Time  in 

Concentration  of 
haemoglobin  (first 

loem  C 

\ogwC0-\ogwCt 

ment 

degrees 

minutes 

sample  =  100  =  C0) 

M-'&10  ° 

t 

1 

60 

0 

100 

2  000 

30 

54 

1.732 

0.0090 

90 

13.5 

1.130 

0.0097 

Mean  0.0094 

2 

62.6 

0 

100 

2.000 

20 

42 

1.623 

O.m 

45 

12 

1.079 

0.020 

70 

4.8 

0.681 

0.019 

Mean  0.019 

3 

65.6 

0 

100 

2.000 

f 

10 

35.5 

1.550 

Q.  045* 

20 

11.0 

1.041 

0.048 

30 

5.0 

0.699 

0.043 

Mean  0.045 

4 

67.6 

0 

100 

2.000 

3 

61.4 

1.788 

6'07i' 

6 

34.8 

1.542 

0.076 

9 

24.9 

1.396 

0.067 

Mean  0.071 

5 

70.4 

0 

100 

2.000 

2 

52.5 

1.720 

o'.ii  " 

4 

25.3 

1.404 

0.15 

6 

14.1 

1.150 

0.15 

7.5 

7.6 

0.886 

0.15 

Mean           0  15 

The  temperature-coefficient  of  the  process  is  seen  to  be  very 
high,  the  velocity-constant  being  multiplied  about  15  times  by 
10  degrees  rise  in  temperature  (from  60-70.4  degrees).  The 
relationship  between  the  temperature  and  the  value  of  K  is  very 
satisfactorily  represented  by  the  Arrhenius  equation : 


a  relation  which,  although  not  strictly  deducible  from  the  gas 
laws  (60)  is  analogous  in  form  to  the  van't  Hoff  equation  con- 
necting the  value  of  the  equilibrium-constant  with  the  tempera- 
ture and  which  is  deducible  from  the  gas  laws.  We  are  probably 
justified  in  concluding,  therefore,  as  we  did  in  the  analogous 
case  afforded  by  the  variation  in  the  dissociation-constant  of 


HEAT,  LIGHT  AND  PRESSURE  307 

oxy haemoglobin  with  temperature,*  that  immediately  prior  to  its 
coagulation  in  the  above  experiments  the  haemoglobin  existed 
in  the  solution  in  a  molecularly  dispersed  condition,  obeying  the 
law  of  Avogadro. 

The  high  temperature-coefficient  of  the  process  explains,  as 
Chick  and  Martin  point  out,  the  apparent,  but  unreal  constancy 
of  the  "coagulation-temperature"  which  was  so  much  insisted 
upon  by  the  earlier  investigators  quoted  above.  With  rising 
temperature  a  point  is  reached  at  which  the  reaction  is  so  rapid 
as  to  appear  almost  instantaneous. 

It  is  obvious  that  the  monomolecular  reaction-formula  would 
apply  either  to  the  dehydration  of  protein  according  to  the  formula 

H2N.R.COH.N.R.COOH  =  HN.R.COH.N.R.CO  +  H20 

I I 

or  to  its  hydration  according  to  a  formula  of  the  type 
Protein  +  H2O  =  coagulated  protein, 

since  in  the  latter  case,  the  mass  of  water  being  very  much  greater 
than  that  of  the  protein,  the  active  mass  of  water  would  be  appre- 
ciably constant  throughout  the  reaction. 

From  the  fact  that  dry  protein,  heated  to  high  temperatures 
does  not  undergo  typical  heat-coagulation,  i.e.,  does  not  lose  its 
solubility  in  water  (24)  Chick  and  Martin  conclude  that  the  heat- 
coagulation  of  protein  is  a  process  of  hydration.  From  the  results 
of  Pauli  and  myself,  cited  in  Chap.  VI,  it  is  evident,  however, 
that  the  process  of  heat-coagulation  is  not  one  of  hydration  but 
of  dehydration  of  the  protein,  f  according  to  some  or  all  of  the 
equations  (i)  to  (iv)  in  Chap.  VI,  section  6.  From  the  fact  that 
the  base-combining  capacity  of  casein  diminishes  with  rising  tem- 
perature, and  also  the  solubility  of  some  of  the  caseinates,  we 
have  concluded  {  that  with  rising  temperature  this  protein,  at 
all  events,  undergoes  some  measure  of  polymerization  on  heating 
through  the  dehydration  of  the  end  —  NH2  and  —  COOH  groups 
of  adjacent  molecules.  §  If  this  were  a  general  phenomenon 

*  Cf.  Chap.  VI.  f  Cf.  also  Michailow  (56)  and  Starke  (98). 

t  Cf.  Chap.  VI  also  1  of  this  chapter  and  T.  Brailsford  Robertson  (83). 

§  Mann  (55)  states  that  in  his  opinion  heat  coagul  tion  is  brought  about 
by  one  portion  of  the  albumin  molecule  precipitating  the  remainder,  a  view 
which  is  essentially  similar  to  that  expressed  above.  Sutherland  (99)  has  also 
expressed  the  view  that  coagulation  of  a  protein  is  the  result  of  polymerization 
through  the  neutralization  of  "Valencies  which  are  usually  latent." 


308  PHYSICAL  PROPERTIES 

then  it  would  follow,  from  van't  HofFs  "Principle  of  Mobile 
Equilibrium"  that  the  hydrolysis  of  protein  (hydration)  is  ac- 
companied by  an  evolution  of  heat,  which  conclusion  is  in  com- 
plete accord  with  experimental  observation.*  The  apparent 
discrepancy  between  the  conclusion  reached  by  Chick  and  Martin 
and  those  which  have  been  formulated  by  other  investigators 
in  this  field  may  therefore  be  due  to  the  fact  that  dry  protein 
has  been  deprived  of  the  elements  of  water  in  its  end  —  NH2 
and  —  COOH  groups,  and  that  these,  consequently,  cannot  react 
to  form  polymers  of  the  protein. f  It  is  evident  that  in  the  process 
of  dehydrating  proteins  by  heat  not  only  the  reaction: 

H2N.R.COH.N.R.COOH  =  HN.R.COH.N.R.CO  +  H20 


occurs,  but  also  reactions  of  the  type 

2  H2N.R.COH.N.R.COOH 

=  H2N.R.COH.N.R.COH.N.R.COH.N.R.COOH  +  H20, 

and  it  is  the  formation  of  these  polymers  which  leads  to  the 
apparently  irreversible  character  of  the  process;  apparently  and 
not  actually  irreversible  because  as  Corin  and  Ansiaux  (22) 
have  shown,  if  a  solution  of  protein  be  cooled  and  vigorously 

*  Cf .  1  and  the  results  of  Wiedemann  and  Ltldeking  cited  in  this  chap- 
ter. The  heat  of  reaction  of  protein  hydrolysis  is  extremely  small;  observers 
using  the  usual  indirect  methods  of  determination  either  failed  to  detect 
any  change  in  the  heat-content  of  the  system  or  else  have  observed  a  very 
slight  disengagement  of  heat  (101),  (49),  (36).  Henderson  and  Ryder,  however, 
using  the  beautiful  and  excessively  sensitive  isothermal  method  of  calorimetry 
of  T.  W.  Richards,  have  demonstrated  that  the  tryptic  hydrolysis  of  casein 
is  accomplished  by  a  progressive  evolution  of  heat  (37).  It  should  be  clearly 
borne  in  mind  in  this  connection  that  the  effect  of  raising  the  temperature  upon 
a  chemical  reaction  is  always  twofold.  It  shifts  the  station  of  equilibrium  in 
one  sense  or  in  the  opposite  and,  always,  it  accelerates  the  reaction  in  either 
sense  to  a  greater  or  less  degree  (i.e.,  it  magnifies  both  velocity  constants,  but 
unequally).  The  action  of  heat  upon  proteins  must  always  be  to  shift  the 
station  of  equilibrium  in  the  direction  of  polymerization  (i.e.,  condensation), 
since  the  reaction  of  hydrolysis  is  exothermic.  But  the  fact  that  fairly  com- 
plete hydrolysis  will  occur  at  the  temperature  of  steam  shows  that  the  shift  in 
equilibrium  between  the  lower  protein  complexes  and  the  amino-acids  which 
are  the  products  of  their  hydrolysis  is  not  so  great  as  to  extinguish  the  reaction 
of  hydrolysis. 

t  It  should  also  be  recollected,  in  this  connection,  that  reactions  occurring 
in  solid  systems  are  notoriously  extremely  slow,  owing  to  the  high  internal 
friction  of  the  system  and  the  consequent  hampering  of  molecular  motion. 


HEAT,  LIGHT  AND  PRESSURE  309 

shaken  just  as  the  first  traces  of  heat  coagulation  appear  the 
incipient  coagula  will  again  pass  into  solution.  The  apparent 
irreversibility  of  the  later  stages  of  heat-coagulation  is  probably 
attributable  to  the  high  internal  friction  of  the  floccula  which 
are  formed,  leading  to  extremely  slow  molecular  movement  and 
the  introduction  of  a  time-element  of  very  considerable  magnitude. 

This  deduction  receives  decided  support  from  the  discovery 
by  Chick  and  Martin  (18)  (19)  (20)  that  the  heat-coagulation  of 
proteins  consists  of  two  processes  which  they  severally  term 
^denaturation "  and  "  agglutination."  In  acid  solutions  the 
process  of  agglutination  or  aggregation  takes  place  at  a  rate 
very  greatly  in  excess  of  denaturation  and  therefore  the  rate  of 
formation  of  flocculated  protein  is  primarily  determined  by  the 
rate  of  denaturation,  i.e.,  by  the  slower  process.  In  alkaline 
solutions,  however,  aggregation  does  not  occur,  but  may  be 
induced  by  subsequent  acidification  or  by  saturation  with  sodium 
chloride.  In  acid  solutions  the  rate  of  denaturation  is  acceler- 
ated by  H+  ions  and  these  decrease  during  the  reaction  through 
the  binding  of  acid  by  the  denatured  protein.  If  the  concen- 
tration of  H+  ions  be  kept  constant  the  reaction  follows  the 
monomolecular  reaction-formula,  but  if  this  precaution  be  omit- 
ted then  the  reaction  velocity  falls  off  more  quickly  than  would 
be  anticipated  from  the  monomolecular  formula  (18)  (100). 
Similarly  in  alkaline  solutions  of  egg-albumin  the  OH'  ion  con- 
centration decreases  during  denaturation,  but  if  the  OH'  concen- 
tration be  kept  constant  the  reaction  is  of  the  first  order. 

The  diminution  in  the  acidity  of  acid  solutions  of  protein 
during  heat-coagulation  has  also  been  observed  and  quanti- 
tatively determined  by  Sorensen  and  Jurgensen  (97)  and  Quagli- 
ariello  (79). 

We  may  conclude,  therefore,  that  the  results  of  Chick  and 
Martin  are  in  satisfactory  harmony  with  the  view  of  Hofmeister 
and  Pauli  that  coagulation,  including  the  heat-coagulation  of 
protein,  is  essentially  a  phenomenon  of  dehydration  of  which 
the  first  stage,  that  of  internal  neutralization  through  the  loss 
of  the  elements  of  water  from  end  —  NH2  and  —  COOH  groups, 
probably  corresponds  to  the  phenomenon  of  "denaturation" 
while  the  subsequent  or  simultaneous  polymerization  of  these 
anhydrides  leads  to  the  formation  of  particles  so  large  as  to 
assume  the  properties  of  matter  in  mass,  i.e.,  flocculi. 


310  PHYSICAL  PROPERTIES 

These  facts  undoubtedly  underlie  and  explain  the  apparent 
transformation  of  serum  albumin  into  serum  globulins  observed 
by  Moll  on  heating  blood  serum  to  which  alkali  had  been  added 
(57).  As  Gibson  (28)  and  Schmidt  (92)  have  pointed  out,  a 
transformation  of  serum  albumin  into  globulin  would  involve  a 
synthesis  of  glycocoll,  which  amino-acid  is  not  contained  in  the 
albumin  molecule.  There  can  be  little  doubt  that  the  protein 
which  Moll  regards  as  globulin  produced  from  albumin  is  in 
reality  " denatured"  albumin  which  acidification  will  flocculate, 
just  as  it  flocculates  a  globulin. 

The  reversible  character  of  "  denaturation "  (presumably 
anhydride-formation  without  polymerization)  is  indicated  by 
the  observation  of  Berczeller  (2)  that  while  the  surface  tension 
of  a  protein  solution  which  is  so  salt-free  as  not  to  coagulate 
on  heating,  nevertheless  diminishes  on  heating,  this  diminution 
is  reversible  and  spontaneously  disappears  on  standing. 

It  has  been  shown  by  Bovie  that  protein  solutions  in  quartz 
vessels  are  coagulated  by  exposure  to  ultraviolet  light  (7).  This 
type  of  coagulation,  like  heat-coagulation,  demonstrably  con- 
sists of  two  separate  processes.  The  first  process,  that  of  denatu- 
ration, has,  like  other  photochemical  processes,  a  very  low 
temperature-coefficient  and  takes  place  almost  as  rapidly  at 
0°  C.  as  at  room  temperature.  The  second  process,  that  of 
agglutination  or  flocculation,  has  a  high  temperature-coefficient 
indicating  that  it  is  not  primarily  a  photochemical  process,  but 
a  spontaneous  consequence  of  the  photochemical  denaturation 
which  precedes  it. 

Fernan  and  Pauli  (25)  have  shown  that  exposure  to  the  radia- 
tions from  radium  leads  to  coagulation  of  protein  (serum  albumin) 
in  acid  or  alkaline  solutions.  Unlike  heat-coagulation  the  co- 
agulation in  acid  solutions  by  radium  radiations  is  accompanied 
by  no  diminution  of  their  H+  concentration. 

It  has  been  shown  by  Bridgman  (9)  that  the  application  of 
very  great  hydrostatic  pressures  results  in  the  coagulation  of 
white  of  egg.  The  pressure  is  applied  very  slowly  to  avoid  any 
rise  in  temperature  due  to  the  compression  and  that  the  effect 
is  not  due  to  heat  is  further  demonstrated  by  the  fact  that  it  is 
more  easily  elicited  at  0  degrees  than  at  20  degrees.  The  applica- 
tion of  five  thousand  atmospheres  produces  stiffening  of  the 
white  of  egg;  six  thousand  atmospheres  compression,  applied  for 


CRYSTALLIZATION  31 1 

thirty  minutes,  produces  an  appearance  of  the  white  resembling 
that  of  curdled  milk,  while  seven  thousand  atmospheres  pressure 
brings  about  complete  gelation. 

5.  The  Crystallization  of  Proteins.  —  A  number  of  proteins, 
particularly  the  vegetable  proteins  and  haemoglobin,  are  readily 
obtainable  in  the  form  of  crystals.  Other  proteins,  such  as 
egg-  and  serum-albumin  only  yield  crystals  with  considerable 
difficulty.  For  the  crystallization  of  these  latter  proteins  mere 
concentration  of  their  solutions  is  insufficient;  inorganic  salts 
in  very  high  concentration  must  also  be  present  (39)  (41)  (42) 
(48)  (94)  (73).  Thus  egg-albumin  may  be  prepared  in  a  crys- 
talline condition  by  adding  to  its  solution  an  equal  volume  of 
saturated  ammonium  sulphate  solution  and  acidifying  with 
acetic  acid.  The  coagulum  which  forms  becomes  crystalline  on 
standing.  It  would  appear  that  the  crystalline  product  is  not 
the  uncombined  protein  but  a  salt  of  the  protein,  formed  with 
an  acid  and  also,  probably,  with  the  ammonium  sulphate  (Cf. 
Chap.  VI)  (59). 

The  protein  crystals  are  optically  true  crystals.  According  to 
Wichmann  all  albumin-crystals  are  either  crystallographically 
identical  or  else  isomorphic.  They  very  readily  absorb  impuri- 
ties (106)  (45)  (95),  and  the  circumstances  of  their  preparation 
involve  the  occlusion  of  a  considerable  quantity  of  the  mother 
liquor.  Hence  repeated  recrystallization  is  required  to  remove 
from  them  even  colloidal  impurities  (95).  Contamination  by 
crystalloidal  substances,  combined  or  physically  associated, 
obviously  cannot  be  avoided,  and  these  must  be  subsequently 
got  rid  of  by  very  prolonged  dialysis  of  the  dissolved  crystals. 

A  monumental  and  most  fundamentally  important  contribu- 
tion to  our  knowledge  of  the  crystallography  of  proteins  has 
been  furnished  by  the  investigations  of  Reichert  and  Brown  (82) 
on  the  relationship  of  the  morphology  of  haemoglobin  crystals 
to  the  biological  classification  of  the  species  from  which  it  is 
derived.  From  an  enormous  number  of  measurements  of  crystal 
angles,  etc.,  conducted  upon  haemoglobins  derived  from  a  large 
variety  of  species,  these  investigators  conclude  in  the  first  place 
that  the  crystals  of  the  species  of  any  genus  belong  to  the  same 
crystallographic  system  and  generally  to  the  same  crystallo- 
graphic  group,  and  they  have  approximately  the  same  axial 
ratios,  or  their  ratios  are  in  simple  relation  with  each  other.  In 


312  PHYSICAL  PROPERTIES 

other  words  the  haemoglobin  crystals  of  any  genus  are  isomorphous. 
In  some  cases  this  isomorphism  may  be  extended  to  include 
several  genera,  but  this  is  usually  not  the  case  unless,  as  in  the 
case  of  the  dogs  and  foxes,  for  example,  the  genera  are  very 
closely  related.  On  the  other  hand  the  oxyhaemoglobin  obtained 
from  the  same  species  always  crystallizes  in  the  same  form,  al- 
though often  with  different  habit  when  obtained  by  different 
methods  of  preparation.  But  upon  comparing  the  haemoglobins 
from  different  species  of  a  genus  it  is  found  that  they  differ  from 
one  another  to  a  greater  or  less  degree  in  angles  or  axial  ratio, 
in  optical  characters  and  particularly  in  those  characters  com- 
prised under  the  general  term  "  crystal  habit,"  so  that  one  species 
can  usually  be  distinguished  from  another  by  the  form  of  its 
haemoglobin  crystals.  But  these  differences,  within  the  limits 
of  a  given  genus,  are  not  such  as  to  preclude  the  crystals  from 
all  species  of  that  genus  being  placed  in  an  isomorphous  series. 

A  clear  relationship  is  thus  seen  to  subsist  between  the  physico- 
chemical  behavior  of  a  constituent  of  organisms  and  their  place 
in  the  phylogenetic  scale  of  relationships  as  established  by  their 
gross  morphology,  and  a  long  stride  has  thus  been  taken  toward 
the  establishment  of  a  physico-chemical  basis  for  morphological 
distinctions.  The  further  and  entirely  independent  question  now 
arises,  however,  as  to  the  chemical  interpretation  of  the  observed 
physico-chemical  phenomena. 

Our  experience  with  the  crystallography  of  inorganic  and  the 
simpler  organic  substances  has  led  us  to  infer  with  a  considerable 
degree  of  confidence  that  substances  which  show  differences  in 
crystallographic  structure  are  different  chemical  substances. 
Crystal  form  is  affected  even  by  isomeric  modifications  which 
analysis,  unaided  by  other  methods  of  investigation,  fails  to 
reveal.  Now  the  enormous  number  of  atoms  in  a  protein  mole- 
cule encourages,  at  first  sight,  the  supposition  that  an  enormous 
and  indeed,  for  all  practical  purposes,  an  infinite  number  of 
isomerides  are  possible  between  which  the  most  refined  methods 
of  analysis  would  not  enable  us  to  distinguish,  but  which  would 
very  probably  differ  from  one  another  in  the  morphology  of 
their  crystals.  In  point  of  fact,  however,  the  available  number 
of  isomers  would  be  very  greatly  restricted  by  the  necessity  of 
maintaining  unaffected  the  amino-acid  groupings  of  the  protein 
moiety  which  could  not  differ  materially  in  different  species 


CRYSTALLIZATION  313 

without  leading  to  decided  differences  in  the  chemical  behavior 
of  the  haemoglobins  which  have  not  been  observed  by  any  in- 
vestigator. Further  doubt  is  thrown  upon  this  interpretation 
of  the  facts  observed  by  Reichert  and  Brown  by  the  observation 
of  Hiifner,  recently  confirmed  with  the  utmost  precision  by 
Butterfield  (13),  Heubner  and  Rosenberg  (38),  and  Schumm  (96) 
that  the  characteristic  absorption  bands  and  the  ratio  of  the 
absorption  of  light  in  different  parts  of  the  spectrum  of  haemo- 
globin  is  absolutely  identical  in  species  so  far  removed  from  one 
another  as  the  horse  and  man  (Schumm)  or  the  rabbit,  sheep 
and  hog  (Heubner  and  Rosenberg).  Now  these  are  properties 
which  we  should  anticipate  might  be  materially  affected  by 
internal  differences  of  atomic  arrangement. 

Further  reason  for  doubting  the  correctness  of  referring  the 
differences  of  crystal  structure  displayed  by  the  haemoglobins  of 
different  animals  to  internal  differences  in  the  molecules  of  the 
haemoglobins  is  supplied  by  the  observation  of  Loeb  and  Brown 
(54)  that  the  crystal-form  of  the  haemoglobin  of  the  mule  is  inter- 
mediate in  character  between  that  of  the  horse  and  that  of  the  donkey. 
For  if  we  assume  that  each  different  crystal  form  represents  a 
different  internal  atomic  arrangement  of  the  haemoglobin  molecule, 
then  the  number  of  such  arrangements,  even  if  very  great,  must 
nevertheless  be  limited.  The  number  of  possible  forms  of  crystals 
must  therefore  also  be  limited  and  moreover  the  possible  modi- 
fications of  forms  must  be  discontinuous,  i.e.,  there  must  exist 
forms  between  which  no  intermediate  forms  are  possible.  This 
being  the  case  it  would  be  very  remarkable  indeed  were  the  hybrid- 
ization of  two  closely  related  species  to  lead  to  the  synthesis 
of  a  new  isomeric  variety  of  haemoglobin  not  yet  appropriated 
by  any  existing  species  of  animal  and  in  addition  lying  between 
the  haemoglobins  of  the  parent  species.  If  analogous  phenomena 
should  be  displayed  by  all  hybrids  and  by  all  varieties  and  mu- 
tations that  might  have  arisen  or  might  conceivably  arise  in  the 
future  we  would  have  to  admit  that  the  haemoglobins  already 
recognizable  as  differing  from  one  another  in  crystal  form  are 
only  a  small  proportion  of  those  which  are  realizable. 

A  much  more  reasonable  supposition  is  that  embodied  in  the 
view  that  the  differences  in  crystal  form  observed  by  Reichert 
and  Brown  are  attributable,  not  to  the  internal  variation  of  atomic 
grouping  in  the  haemoglobin  molecules  but  to  external  varia- 


314  PHYSICAL  PROPERTIES 

tions  in  the  milieu  from  which  they  are  crystallized.  The  tech- 
nique adopted  by  Reichert  and  Brown  was  to  induce  crystal- 
lization directly  in  the  laked  blood.  Now  we  know  from  the 
observations  of  the  immunologists  that  the  blood  plasma  from 
any  species  of  animal  differs  antigenically  from  that  derived 
from  any  other  species  (62)  and,  since  all  known  antigens  are 
proteins,  we  infer  that  the  proteins,  or,  more  probably,  the  com- 
pound protein  complexes  (Cf.  Chap.  VII)  in  blood  plasmas 
derived  from  different  species  are  in  certain  definite  respects 
different  from  each  other.  The  crystals  of  each  species  studied 
by  Reichert  and  Brown  were  therefore  deposited  from  a  different 
medium  and  it  is  not  improbable  that  the  observed  differences 
in  the  crystals  are  attributable  to  these  known  differences  in 
the  media  in  which  they  were  formed.  It  is  well  known  that 
crystal-habit  is  modified  by  alterations  of  the  medium  from  which 
the  crystals  are  deposited.  That  modifications  of  this  origin  so 
great  as  to  preclude  inclusion  of  the  crystals  formed  in  different 
media  in  the  same  isomorphous  series  have  not  hitherto  been 
observed  in  the  domain  of  inorganic  chemistry  is  not  improbably 
attributable  to  the  simpler  character  of  the  conditions  accom- 
panying crystallization  in  inorganic  or  non-colloidal  media.  We 
have  seen  that  there  are  many  reasons  for  supposing  that  proteins, 
even  in  solution,  are  disposed  in  a  certain  reticular  structure 
and  if,  as  the  facts  dwelt  upon  in  Chap.  VII  would  seem  to  in- 
dicate, characteristic  protein  complexes,  formed  by  the  union 
in  differing  proportions  of  a  relatively  small  number  of  simpler 
protein  components,  exist  in  each  type  of  blood  plasma  we  may 
well  suppose  that  the  reticular  structures  of  the  solutions  com- 
prising these  plasma  would  likewise  differ  from  one  another. 
Having  regard  to  the  markedly  cohesive  properties  of  proteins, 
crystallization  within  the  meshes  of  such  a  reticulum  might 
very  conceivably,  through  external  strains  imposed  by  points 
of  attachment  to  the  reticulum,  modify  the  effects  of  the  internal 
strains  which  find  their  expression  in  crystal  form. 

This  hypothesis  finds  decided  support  in  the  fact  first  observed 
by  Halliburton  (39)  (31)  and  confirmed  by  Reichert  (81)  that 
the  crystal  form  of  oxyhaemoglobin  derived  from  a  given  species 
may  be  profoundly  modified  by  admixture  with  the  blood  of 
another  species.  The  following  are  illustrative  results  obtained 
by  Halliburton,  the  " normal"  form  of  rat  haemoglobin  crystals 


CRYSTALLIZATION 


315 


being  rhombic,  those  obtained  from  guinea-pigs  being  normally 
tetragonal  and  those  from  squirrel's  blood  hexagonal. 


Blood  of 

Mixed  with  that  of 

Form  of  haemoglobin  crystals  deposited  from  the  mixture 

Rat 

Squirrel 

Both  rhombic  prisms  and  hexagons  present. 

Rat 

Guinea-pig 

No  rhombic  prisms  of  the  shape  usually  seen 
in  rat's  blood  present.  No  tetrahedra. 
Crystals  are  all  rhombic  prisms  with  hexa- 
gonal habit. 

Squirrel 

Guinea-pig 

Hexagonal  plates  and  tetrahedra  both  pres- 
ent. Many  tetrahedra  imperfect.  The 
tetrahedra  all  reduced  to  about  half  the 
size  of  those  prepared  from  the  unmixed 
blood  of  the  same  guinea-pigs. 

Dog 

Squirrel 

Fine  rhombic  needles  and  hexagonal  plates 
both  present  in  abundance. 

Dog 

Guinea-pig 

The  greater  number  of  the  crystals  formed 
are  very  small  tetrahedra  about  a  quarter 
the  size  of  those  prepared  from  the  blood  of 
the  same  guinea-pigs.  The  optical  prop- 
erties are,  however,  the  same.  Rhombic 
prisms,  very  slender,  like  those  of  dog's 
blood,  are  also  seen. 

According  to  Reichert  the  degree  of  modification  of  crystal 
form  induced  by  admixture  of  two  bloods  depends  very  greatly 
upon  the  proportion  in  which  they  are  mixed. 

In  view  of  these  facts  there  can  be  little  doubt  that  the  nature 
of  the  milieu  in  which  crystallization  occurs  does  play  an  im- 
portant part  in  determining  the  form  of  the  crystals  which  are 
deposited,  and  having  regard  to  the  known  individuality  of  the 
plasma  from  different  biological  species  it  would  appear  unneces- 
sary to  seek  further  for  the  origin  of  the  differences  in  crystal 
form  of  the  oxyhaemoglobins  derived  from  blood  of  different 
species  of  animals. 

We  are  now  in  a  position,  also,  to  interpret  the  changes  in 
crystal-form  which  result  from  repeated  re-crystallization  of 
haemoglobin  (Halliburton  (30)  (31)),  for  as  Wichmann  (106)  and, 
more  recently,  Katz  (45)  have  shown,  the  crystalline  proteins 
swell  in  or  absorb  the  surrounding  fluid  menstruum  in  a  manner 
analogous  to  the  swelling  of  jellies.  A  number  of  re-crystal- 
lizations are  therefore  required  to  remove  completely  traces  of 
the  original  menstruum  in  which  crystallization  occurred. 


316  PHYSICAL  PROPERTIES 

Bradley  and  Sansum  (8)  believe  that  the  haemoglobins  from 
different  animals  are  antigenically  different  because  guinea-pigs 
sensitized  to  ox  or  dog  haemoglobin  failed  to  display  anaphylactic 
shock  or  reacted  but  slightly  to  haemoglobins  of  other  origins, 
while  they  reacted  strongly  to  the  haemoglobin  with  which  they 
were  sensitized.  As  the  haemoglobin  preparations  employed  by 
Bradley  and  Sansum  were  admittedly  (with  the  exception, 
they  believe,  of  the  dog  haemoglobin)  not  free  from  contamina- 
tion by  serum  the  interpretation  of  these  results  is  open  to  serious 
question.  Doubt  is  especially  thrown  upon  this  evidence  for 
the  specificity  of  haemoglobins  from  different  species  by  the  fact 
that  the  animals  sensitized  to  the  purest  preparation  of  haemo- 
globin employed,  that  of  the  dog,  reacted  strongly  not  only  to 
to  dog  haemoglobin  but  also  to  dog  serum.  Having  regard  to  the 
investigations  of  Wichmann  and  Katz,  cited  above,  revealing 
the  marked  ability  of  crystalline  proteins  to  absorb  the  menstruum 
from  which  they  are  deposited,  and  to  the  observation  of  Schulz 
and  Zsigmondy  (95)  that  egg  albumin  must  be  recrystallized 
from  3  to  6  times  in  order  to  remove  appreciable  contamination 
by  other  proteins,  we  may  infer  that  in  all  probability  the  speci- 
ficities demonstrated  by  Bradley  and  Sansum  are  serum-speci- 
ficities and  not  haemoglobin-specificities. 

According  to  Howell  (43),  fibrin  must  now  be  added  to  our 
list  of  crystallizable  proteins.  He  finds  that  when  fibrinogen  is 
precipitated  by  thrombin,  the  fibrin,  in  media  of  normal  H+ 
concentrations,  separates  out  in  crystalline  needles  readily  recog- 
nizable as  such  under  the  ultramicroscope.  They  vary  in  length 
from  10  to  30  microns  and  form  a  close  mesh- work.  The  normal 
blood-clot  is  therefore  a  crystalline  gel.  The  blood  of  inverte- 
brates, however,  yields  a  non-crystalline  gel  and  a  similar  gel  is 
yielded  by  mammalian  fibrin  in  alkaline  media.  Such  non- 
crystalline  gels,  however,  fail  to  display  the  spontaneous  con- 
traction or  "synaeresis"  which  is  so  characteristic  of  normal 
blood-clots. 

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(67)  Ostwald,  Wo.,  "Grundriss  der  Kolloidchemie,"  2te  Aufl.,  Dresden, 

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(68)  Paal,  C.,  Ber.  d.  d.  chem.  Ges.,  25  (1892),  p.  1202. 

(69)  Palme,  H.,  Zeit.  f.  physiol.  Chern.,  92  (1914),  p.  177. 

(70)  Pauli,  W.,  Arch.  f.  Exper.  Path,  und  Pharm.,  36  (1895),  p.  100. 

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(79)  Quagliariello,  E.,  Biochem.  Zeit.,  44  (1912),  p.  157. 

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CHAPTER  XIII 


CERTAIN  PHYSICAL  PROPERTIES  OF  PROTEIN  SOLUTIONS,  ETC. 

1.  The  Viscosity  of  Protein  Solutions.  —  Protein  solutions  are 
usually  characterized  by  the  possession  of  a  high  viscosity.  The 
question  has  been  raised  whether  the  viscosity  is  similar  in  nature 
to  that  of  a  solution  of  a  crystalloid  substance,  or  whether  it  is 
not,  rather,  comparable  with  the  viscosity  of  a  suspension  of  solid 
particles  (40).  In  support  of  this  latter  view  it  has  been  pointed 
out  that  Bottazzi's  measurements  of  the  influence  of  concen- 
tration upon  the  viscosity  of  protein  solutions  indicate  that  the 
viscosity  of  these  solutions  does  not  change  continuously  with 
change  in  concentration,  as  it  does  in  solutions  of  crystalloids,  but 
changes  per  saltum  (12).  The  measurements  of  Bottazzi  were 
made  upon  relatively  few  and  widely  separated  concentrations, 
however,  and  subsequent  observers  have  not  confirmed  his  results 
(108)  (59).  Thus  Sackur  finds  that  the  viscosity  of  solutions  of 
the  "basic"  casemates  of  sodium  and  ammonium  (i.e.,  containing 
about  80xlO~5  equivalents  of  base  per  gram  and  neutral  to 
phenolphthalein)  varies  with  the  concentration  according  to  the 

Arrhenius-Euler  formula  —  =  An,  where  rj  is  the  viscosity  of  the 

*7o 

solution,  T]Q  that  of  the  solvent,  n  is  the  concentration  of  the  solu- 
tion and  A  is  a  constant,  the  numerical  value  of  which  depends 
upon  the  nature  of  the  dissolved  substance  and  upon  the  tempera- 
ture (2).  The  following  are  Sackur's  results: 


n  (in  equivalent) 
sodium) 

-2  (15  degrees) 

70 

log  A 

0.01830 

1.870 

14.8 

0.01370 

1.581 

14.5 

0.00915 

1.363 

14.3 

0.00547 

1.202 

14.6 

0.00458 

1.165 

14.5 

A  remarkable  feature  of  these  results  is  the  extraordinarily  high 
value  of  A,  involving  a  very  rapid  increase  in  viscosity  with 

320 


VISCOSITY 


321 


increasing  concentration.  For  the  majority  of  crystalloids  the 
value  of  A  is  not  greatly  in  excess  of  unity,  while  for  sodium 
casemate,  as  we  see,  it  is  of  the  order  of  1014.  This  fact  alone 
would  lead  us  to  suspect  that  the  mechanism  which  produces 
the  viscosity  of  these  solutions  is  different  in  nature  from  that 
which  produces  the  viscosity  of  solutions  of  crystalloids.  Sackur 
has  endeavored  to  ascertain  which  constituent  of  the  solutions  of 
sodium  caseinate  plays  the  greater  part  in  determining  their 
viscosity.  He  arrived  at  a  conclusion  the  correctness  of  which 
more  recent  investigations  have  fully  established,  by  a  process  of 
reasoning,  however,  which  recent  investigations  have  shown  to 
be  in  some  respects  fallacious.  He  argued  that  the  viscosity  of 
these  solutions  might  be  attributable,  primarily,  to  undissociated 
sodium  caseinate,  or  the  product  of  its  hydrolytic  dissociation,* 
i.e.,  free  casein,  or  to  caseinate  ions.  Hydrolytic  dissociation 
would,  he  believed,  be  diminished  by  the  addition  of  alkali  and 
increased  by  the  addition  of  acid.  In  the  former  case,  according 
to  his  view,  the  number  of  caseinate  ions  should  be  increased,  in 
the  latter  decreased.  He  found  that  the  addition  of  alkali  in- 
creased and  the  addition  of  acid  diminished  the  viscosity  of  a  neutral 
(to  phenolphthalein)  solution  of  sodium  caseinate;  the  following 
are  among  his  results: 


Per  cent  of  casein 

Normality  of  total 
sodium 

-  (15  degrees) 
fo 

0.716 

0.0063 

1.24 

0.716 

0.0126 

1.36 

0.716 

0.0189 

1.38 

0.716 

0.0252 

1.34 

hence,  he  argued,  the  viscosity  of  these  solutions  is  primarily 
attributable  to  caseinate  ions. 

We  have  seen,  however  (Chaps.  IV  and  VIII),  that  the  casemates 
do  not  undergo  hydrolytic  dissociation  in  solution.  In  fact  the 
initial  solution  to  which  Sackur  added  alkali  in  order  to  suppress 
hydrolytic  dissociation  was  neutral  to  phenolphthalein  and  there- 
fore could  not  have  contained  more  than  10~5  N  NaOH  derived 
from  hydrolytic  dissociation  of  the  sodium  caseinate.  We  have 
also  seen  (Chap.  IX)  that  on  adding  alkali  to  a  solution  of  a  casein- 

*  Since  free  casein  is  insoluble  this  possibility  may  be  dismissed. 


322  PHYSICAL  PROPERTIES 

ate  which  is  neutral  to  phenolphthalein  the  added  alkali  does  not 
remain  unneutralized,  as  Sackur  assumes,  but,  on  the  contrary, 
is  partially  bound  by  the  casein  until  180xlO~5  equivalents  are 
combined  with  every  gram  of  casein.  But  we  have  also  seen 
(Chap.  IX)  that  each  successive  equivalent  of  combined  alkali 
splits  another  —  NHOC—  bond  in  the  casein  molecule  and  con- 
sequently gives  rise  to  another  pair  of  ions.  With  increasing 
content  of  combined  base,  therefore,  the  caseinate,  apart  from 
slight  modifications  of  its  degree  of  dissociation,  yields  a  corre- 
sponding proportion  of  ions.  The  fact  that  the  viscosity  of 
caseinate  solutions  markedly  increases  with  alkalinity  is  therefore 
in  strong  support  of  Sackur's  thesis  that  the  viscosity  of  these 
solutions  is  primarily  attributable  to  protein  ions,  although  not 
for  the  reasons  which  he  advances.  The  fact  that  the  increase  in 
viscosity  with  increasing  alkalinity  attains  a  maximum  at  just 
about  the  same  time  that  the  combining  capacity  of  the  casein 
attains  a  maximum  (Cf .  above  table  and  the  tables  and  diagram 
in  Chap.  IX)  lends  very  striking  support  to  this  hypothesis. 

The  view  that  the  viscosity  of  protein  solutions  is  in  a  remark- 
ably high  degree  dependent  upon  the  protein  ions  which  they 
contain  has  also  been  advanced,  with  substantial  experimental 
support,  by  W.  B.  Hardy  (41)  and  by  Bottazzi  (14).  The  latter 
observer  has  shown  that  the  viscosity  of  protein  solutions  is  at  a 
minimum  when  ionic  protein  is  absent,  when  the  protein  is  un- 
combined  with  base  or  acid,  and  that  on  adding  either  acids  or 
bases  to  this  solution  the  viscosity  increases. 

Now  a  very  little  consideration  suffices  to  show  that  the  viscosity 
of  protein  solutions  is  of  a  different  type  from  the  viscosity,  for 
example,  of  solutions  of  sugar  or  glycerol  in  water.  Apart  from 
the  extraordinary  magnitude  of  A,  alluded  to  above,  the  type  of 
viscosity  exhibited  by  solutions  of  proteins  differs  from  the  vis- 
cousness  of  a  glycerol-water  mixture  in  that  it  affords  no  hindrance, 
or  very  slight  hindrance,  to  the  motion  of  ions  and  of  crystalloid 
molecules.  The  properties  of  agar  are,  in  this  respect,  very  similar 
to  those  of  protein.  Thus  Graham  (35)  showed  that  the  velocity 
with  which  crystalloids  diffuse  through  gelatin  jellies  is  remarkably 
near  to  that  with  which  they  diffuse  through  water,  and  Voigt- 
lander  (126)  has  confirmed  this  result  for  agar  jellies.  Similar 
results  have  been  obtained  by  Hiif ner  (47) .  According  to  Bechhold 
and  Ziegler  (6)  the  rate  of  diffusion  of  (at  any  rate  concentrated) 


VISCOSITY  323 

crystalloids  is  diminished  by  gelatin  jellies,  and  the  degree  of 
hindrance  is  materially  modified  by  the  presence,  within  the  jelly, 
of  other  dissolved  substances,  but  the  hindrance  is  extremely  small 
in  comparison  with  the  enormous  viscosity  of  the  jellies. 

Similarly  Reformatzky  (90)  has  shown  that  the  velocity  with 
which  methyl  acetate  is  decomposed  by  acids  in  agar  jelly  (i.e.,  the 
number  of  molecular  collisions  per  second)  is  within  1  per  cent  of 
its  value  in  pure  water. 

Lodge  (65),  Whetham  (129)  (130)  and  Masson  (69)  have  shown 
that  the  specific  mobilities  of  the  majority  of  inorganic  ions  is  the 
same  in  agar  jellies  as  it  is  in  water.  Dumanski  (28)  has  shown 
that  if  allowance  be  made  for  the  diminution  in  the  cross-section 

of  the  conducting  field,  due  to  the  presence  of  gelatin  (  =  (-)  » 
where  g  is  the  number  of  grams  of  gelatin  per  gram  of  solution  and 
c  the  specific  gravity  of  gelatin) the  conductivities  of  inorganic 

salt  solutions  in  gelatin  jellies  are  very  slightly  less  than  those  of 
equally  concentrated  solutions  in  pure  water. 

We  have  seen*  that  the  dependence  of  the  conductivity  of 
protein  solutions  upon  their  dilution  is  of  perfectly  normal  char- 
acter, resembling  the  dependence  of  the  conductivity  of  a  crystal- 
loidal  electrolyte  upon  dilution,  despite  the  fact  that  the  protein 
solutions,  as  the  above-cited  results  of  Sackur  reveal,  vary  enor- 
mously in  their  viscosity  with  dilution. 

On  the  other  hand,  the  intimate  dependence  of  the  conductivity 
of  solutions  of  electrolytes  upon  the  ordinary  type  of  viscosity  has 
been  commented  upon  and  quantitatively  estimated  by  a  host  of 
observers,  among  whom  Walden  may  be  especially  mentioned 
(127)  (51)  (37).  Viscosities,  very  much  less  than  those  of  the 
dilutest  jellies,  profoundly  diminish  the  conductive  power  of 
electrolytes.  Nor  must  it  be  imagined  that  the  viscosity  of  a 
protein  jelly  is  essentially  different,  in  any  respect  save  magnitude, 
from  that  of  a  protein  solution,  for,  as  von  Schroeder  (110)  has 
shown,  the  viscosity  of  a  solution  of  gelatin,  cooled  below  the 
gelation-point,  increases  progressively  and  regularly  with  time, 
until  the  extremely  viscous  solution  passes  insensibly  into  a  jelly. 

Not  only  inorganic,  but  also  protein  ions  are  profoundly  in- 
fluenced in  their  mobilities  by  the  type  of  viscousness  which 

*  Cf.  Chap.  X,  also  W.  B.  Hardy  (41). 


324  PHYSICAL  PROPERTIES 

alcohol-  or  glycerol-water  mixtures  exhibit.  Thus  we  have  seen, 
in  Chapter  XI,  that  the  mobilities  of  caseinate  ions  in  alcohol- 
water  mixtures  are  almost  exactly  in  inverse  proportion  to  the 
viscosity  of  the  solvent,  and  this  despite  the  fact  that  the  viscosity 
of  the  solvent,  measured  by  the  time  which  it  takes  to  run  through 
a  capillary  tube,  is  so  profoundly  influenced  by  the  introduction  of 
the  caseinate  itself  as  to  be  in  many  cases,  doubled  in  magnitude. 
Solutions  of  KC1  in  alcohol-water  mixtures  are,  comparatively 
speaking,  unaltered  in  their  viscousness  by  the  KC1,  yet  the 
dependence  of  the  conductivities  of  solutions  of  potassium  casein- 
ate  upon  the  percentage  (between  0  and  60  per  cent)  of  alcohol 
which  they  contain  obeys  exactly  the  same  law  as  that  which 
applies  to  the  conductivity  of  KC1  solutions  in  alcohol-water 
mixtures,  a  law  which  implicitly  involves  the  conclusion  that  the 
total  effect  is  due  solely  to  the  alteration  in  the  mobilities  of  the 
ions  attributable  to  the  viscousness  of  the  solvent.  To  make 
the  matter  clear  by  reference  to  a  numerical  example:  Referring  to 
the  tables  in  Chapter  XI  we  see  that  the  viscosity  of  JV/10  KOH 
(dilute  alkali)  is  doubled  by  the  introduction  of  3.125  per  cent  of 
casein;  halving  the  concentration  of  the  solution,  according  to 
Sackur's  results  cited  above,  must  more  than  halve  the  effect  of  the 
caseinate  upon  its  viscousness;  yet  the  mobilities  of  the  caseinate 
ions  are  unaltered  by  this.  The  viscosity  of  the  solvent  can  be 
also  doubled  by  the  introduction  of  40  per  cent  alcohol  and  this, 
on  the  contrary,  halves  the  mobility  of  the  caseinate  ions.  The 
alcohol  considerably  modifies  the  degree  to  which  the  caseinate 
alters  the  viscousness  of  the  solvent,  yet  this  fact  does  not  at  all 
disturb  the  regularity  of  the  relation  between  the  viscosity  of  the 
solvent  and  the  mobility  of  the  caseinate  ions  which  are  dissolved 
therein.  In  estimating  the  influence  of  viscosity  upon  the  mobilities 
of  caseinate  ions  we  can  entirely  disregard  that  portion  of  the  viscosity 
of  the  solution  which,  although  comparable  in  magnitude  with  the 
viscosity  of  the  solvent,  is  attributable  to  the  caseinate  itself. 

Bearing  in  mind  the  possibility,  which  was  indicated  in  the 
previous  chapter,  that  protein  solutions  may  contain  a  pre- 
formed molecular  structure  analogous  to  that  of  the  jellies  or 
coagula  which  they  can  form,  we  are  strongly  impelled  towards 
the  belief  that  the  type  of  viscosity  which  solutions  of  proteins 
exhibit  may  in  some  manner  owe  its  existence  to  this  structure, 
and  not  to  the  type  of  internal  friction  which  hinders  molecular 


VISCOSITY  325 

and  ionic  motion.  Thus  a  netlike  structure,  such  as  a  tennis  net, 
will  offer  no  hindrance  to  the  passage  through  it  of  a  quickly 
moving  body  which  is  smaller  than  its  meshes,  other  than  that 
which  is  due  to  the  fact  that  the  material  which  composes  the  net 
occupies  a  small  fraction  of  the  area  which  the  body  must  traverse, 
but  to  any  force  which  involves  deformation  of  the  structure,  for 
instance,  a  force  which  seeks  to  drag  it  through  a  small  tube,  it  will 
offer  a  very  considerable  resistance.  On  the  other  hand  the 
resistance  which  is  offered  to  a  small  moving  body  by  a  viscous 
liquid  (viscous,  that  is,  in  the  ordinary  sense)  is  accurately  meas- 
ured by  the  resistance  which  the  liquid  offers  to  passage  through 
a  tube.  Now  the  direct  methods  which  we  employ  to  measure 
the  viscosity  of  fluids  are  all  such  (rotation  of  a  relatively  large 
body  within  the  fluid;  passage  of  the  liquid  through  a  capillary 
tube,  etc.)  as  would  involve  the  deformation  of  any  molecular 
structure  within  the  fluid.  Our  direct  methods  of  estimating 
viscosity  do  not  enable  us  to  distinguish  between  that  type  of 
viscosity  which  is  attributable  to  a  structure  within  the  fluid  and 
the  other  type  of  viscosity  which  we  may  term  "true"  internal 
friction.  The  indirect  measurement  which  the  conductivity  esti- 
mations afford,  however,  only  reveals  the  latter  type  of  viscosity 
and,  as  we  have  seen,  when  we  employ  this  method  of  estimation 
the  presence  of  protein  is  found  to  leave  the  viscosity  of  the  solvent 
unaltered.* 

If  we  admit,  however,  that  the  viscosity  of  protein  solutions  is 
due  to  a  molecular  net-structure  within  them,  then  we  are  forced 
to  a  conclusion  which,  in  the  light  of  our  present  inadequate 
knowledge  of  the  mechanics  of  ionization,  appears  very  curious. 
We  have  seen  (Cf.  above)  that  the  viscosity  of  protein  solutions 
must  be  attributable,  primarily,  to  the  protein  ions  which  they 
contain.  We  are  led  to  conclude,  therefore,  that  the  net-structure 

*  Save  to  the  degree  involved  by  the  fact  that  the  material  composing  the 
net-structure  occupies  a  certain  proportion  of  the  cross-area  of  the  conducting 
field.  As  we  have  seen  above,  the  results  of  Dumanski  show  that  if  allowance 
be  made  for  this  diminution  in  cross-area  the  conductivities  of  solutions  of 
electrolytes  in  gelatin  jellies  are  nearly  identical  with  those  of  equally  concen- 
trated solutions  in  water.  The  slight  deviations  which  Dumanski  observed 
are  probably  attributable  to  the  fact  that  the  calculation  of  this  diminution  in 
area  involved  the  specific  gravity  of  the  gelatin  in  its  dissolved  condition,  and 
this  is  not  necessarily  the  same  as  that  of  unhydrated  gelatin,  i.e.,  gelatin  in 
its  undissolved  condition. 


326  PHYSICAL  PROPERTIES 

within  protein  solutions  is  built  up  out  of  protein  ions.*  Such 
a  conclusion  is  totally  out  of  harmony  with  the  prevailing  view 
that  ions,  in  solutions  of  electrolytes,  are  mutually  independent 
and  physically  discrete  bodies,  and  would  appear  to  invite  a  dis- 
tinction between  the  mode  of  ionization  of  ordinary  electrolytes 
and  that  of  protein  salts.  It  is  very  questionable,  however, 
whether  such  a  distinction  would  be  valid.  Many  modern  in- 
vestigations point  to  the  existence  of  some  modification  of  the 
familiar  "  Grotthus-chain "  in  solutions  of  electrolytes,  most 
impressive  among  which  is  possibly  the  fact  that  in  watery  solu- 
tions the  most  rapidly-moving  ions  are  those  which,  by  their 
combination,  give  rise  to  water  itself,  i.e.,  hydrogen  and  hydroxyl, 
while  in  other  solvents  hydrogen  and  hydroxyl  are  not  the  most 
rapidly  moving,  but  those  which,  by  their  combination,  give  rise  to 
the  solvent  (24)  (125)  (25)  (60).  The  fact  that  the  viscosities  of 
solutions  of  crystalloid  electrolytes  have  not  so  far  revealed  the 
presence  of  a  net-structure  within  them  is  possibly  attributable  to 
the  tenuity  of  the  net  and  to  the  fineness  of  its  framework;  to 
revert  to  the  analogy  employed  above,  a  net  of  the  finest  and  most 
flexible  silk  will  readily  pass  without  appreciable  resistance 
through  a  tube  which  would  offer  a  considerable  resistance  to  the 
passage  of  a  net  of  coarse  thread.  The  phenomena  to  which  I 
have  drawn  attention  may  quite  conceivably  be  of  very  general 
occurrence,  and  of  greater  physical  magnitude  in  solutions  of 
proteins  simply  because  of  the  greater  size  of  the  protein  mole- 
cules. In  this  connection  the  views  regarding  the  nature  of 
ionization  which  have  been  put  forward  by  Wm.  Sutherland  (118) 
offer  extremely  tempting  suggestions. 

The  hypothesis  that  gelatin  solutions,  even  those  which  do  not 
gelatinize,  contain  a  structure  has  also  been  put  forward  by  Wo. 
Ostwald  (80).  The  influence  of  added  inorganic  salts  upon  the 
viscosity  of  gelatin  solutions  has  been  studied  by  von  Schroeder 
(loc.  cit.);  this  observer  finds  that  the  chlorides  and  nitrates  of 
the  alkalies  diminish  the  viscosity  of  gelatin  solutions  while  sul- 
phates increase  it.  Moreover  the  alteration  in  viscosity  induced 
by  the  added  salts  does  not  vary  continuously  in  a  definite  sense 

*  Indeed,  whether  we  assume  that  the  viscosity  of  protein  solutions  is  due 
to  protein  ions  or  not,  it  would  be  difficult  to  avoid  this  conclusion  in  the  light 
of  the  fact  that  many  highly  viscous  solutions  of  casemates  are  over  90  per  cent 
dissociated  (Cf.  Chap.  X). 


VISCOSITY  327 

as  more  of  the  salt  is  added,  but  the  curve  displaying  the  influence 
of  the  concentration  of  the  salt  upon  the  viscosity  of  the  solution 
exhibits  very  marked  maxima  and  minima.  Now  Ostwald  has 
found  that  the  influence  of  added  salts  upon  the  swelling  of  gelatin 
plates  in  water  (Cf.  Chap.  XII)  is  of  a  similar  character  to  their 
influence  upon  the  viscosity  of  gelatin  solutions;  that  the  degree 
of  swelling  runs  closely  parallel  with  the  diminution  in  the  viscosity 
of  solutions,  as  determined  by  von  Schroeder,  since  the  concen- 
trations of  salts  at  which  maxima  of  swelling  occur  are  nearly  identi- 
cal with  those  at  which  minima  in  the  viscosity  of  solutions  are 
observed.  From  this  Ostwald  argues  that  the  passage  of  gelatin 
into  solution  does  not  destroy  the  structure  of  the  gel  but  that  this 
structure  persists  in  solution. 

Sutherland  (119)  in  developing  the  theories  to  which  I  have 
referred  above,  has  also  expressed  the  opinion  that  protein  solu- 
tions possess  a  structure.* 

Of  great  interest  in  this  connection  is  the  observation  which  a 
number  of  investigators  have  made  (110)  (59)  (34)  (109)  (33)  that 
the  viscosities  of  gelatin  solutions,  insufficiently  concentrated  to 
gelatinize,  change  somewhat  on  standing.  Garrett  has  also 
observed  (33)  that  the  logarithmic  decrement  of  a  disc  oscillating 
in  gelatin  or  albumin  solutions  is  not  a  constant,  as  it  is  in  water 
or  other  homogeneous  fluids,  but,  on  the  contrary,  increases  as  a 
linear  function  of  the  time.  This  was  traced  to  adhesion  between 
the  disc  and  the  protein;  if  the  disc  was  taken  out  of  the  fluid  and 
washed  the  initial  value  of  the  decrement  was  always  the  same. 
This  is  obviously  to  be  explained,  on  the  basis  of  the  above 
hypothesis,  by  the  adhesion  of  portions  of  the  net-structure  to 
the  oscillating  disc.  Garrett  has  further  observed  that  very 
slight  mechanical  disturbance  of  a  gelatin  solution  produces  con- 
siderable alteration  in  the  magnitude  of  the  oscillation-decrement. 

Preliminary  heating  diminishes  the  viscosity  of  gelatin  solutions 
and  the  magnitude  of  the  effect  is  a  function  of  the  time  of  expo- 
sure to  the  higher  temperature  (110)  (33).  Garrett  attributes  this 
phenomenon  to  a  partial  destruction  by  heating  of  a  structure 
within  the  fluid.  Ostwald  (79)  has  found  that  preliminary 
heating  of  gelatin  increases  the  rapidity  with  which  it  swells  in 
water.  He  also  interprets  this  by  supposing  that  the  structure  of 

*  Cf.  also  C.  O.  Weber  (128). 


328  PHYSICAL  PROPERTIES 

the  gelatin  is  partially  destroyed  by  heating.  Garrett  has  also 
found  that  in  a  solution  of  gelatin  which  had  been  boiled  the 
logarithmic  decrement  of  an  oscillating  disc  does  not  increase  with 
time. 

According  to  Lichtwrtz  and  Renner  (61)  the  viscosity  of  serum 
albumin  solutions  falls  with  rising  temperature  from  15  to  60°  C. 
in  the  same  proportion  as  the  viscosity  of  water.  Chick  and 
Lubrzynski  (20)  have  also  shown  that  the  influence  of  tempera- 
ture upon  the  viscosity  of  egg-albumin  solutions  containing  up  to 
20  per  cent  of  protein  is  slight  and  a  linear  function  of  temperature 
being  similar  to  the  influence  of  temperature  upon  the  viscosity 
of  a  solution  of  a  crystalloid.  In  solutions  containing  a  higher 
percentage  of  protein,  however,  the  relationship  between  viscosity 
and  temperature  is  curvilinear,  the  decrease  being  most  rapid  at 
low  temperatures.  From  these  results  it  would  appear  reasonable 
to  infer  that  a  change  of  temperature  at  temperatures  lying  below 
those  necessary  to  induce  coagulation  affects  the  viscosity  of  a 
protein  solution  chiefly  through  altering  the  viscosity  of  the  solvent, 
a  deduction  which  is  in  harmony  with  the  view  expressed  above 
that  proteins  in  solution  are  not  in  any  large  proportion  present 
therein  in  the  form  of  independently  mobile  particles. 

It  has  been  shown  by  Christiansen  (22)  that  acid  protein  solu- 
tions attain  a  maximum  of  viscosity  when  a  slight  excess  of  free 
acid  is  present,  presumably  coinciding  approximately  with  the 
attainment  of  maximum  combining  capacity  for  the  acid.  The 
acidity  which  produces  maximum  viscosity  is  also  the  optimum 
acidity  for  the  digestive  action  of  pepsin.  The  viscosity  of  the 
solution  decreases  during  digestion,  a  fact  which  had  previously 
been  demonstrated  by  Spriggs  (116). 

2.  The  Cohesiveness  of  Protein  Solutions.  —  Closely  allied 
to  the  property  of  viscosity  is  the  property  which  may  be  somewhat 
vaguely  designated  "cohesiveness."  The  influence  of  a  variety  of 
aqueous  solutions  of  acids,  bases  and  salts  upon  the  cohesiveness  of 
gluten  has  been  investigated  by  Wood  and  Hardy  (132).  These 
observers  prepared  gluten  by  washing  flour  in  a  stream  of  water 
to  remove  the  starch.  The  protein  residue  is  a  coherent  stringy 
mass,  insoluble  in  water  and  consisting  essentially  of  a  mixture  of 
gliadin  and  glutenin  (Cf.  Chap.  II).  The  action  of  solutions  upon 
the  cohesiveness  of  the  gluten  was  estimated  quantitatively  by 
suspending  a  small  mass  of  gluten  on  a  bent  glass  rod  in  a  beaker 


ELASTICITY  329 

containing  120  cc.  of  the  solution.  It  was  found  that  dilute  acids 
very  quickly  led  to  a  very  marked  diminution  in  cohesiveness, 
allowing  the  drop  to  fall  off  the  rod  and  disperse,  forming  a  cloudy 
solution.  More  concentrated  acid,  however,  maintains  the  cohe- 
sion; in  other  words,  with  increasing  concentration  of  acid  the 
cohesiveness  of  the  gluten  is  first  lowered,  until  it  reaches  a  mini- 
mum, and  then  raised  until  it  regains  its  value  in  water.  There 
is  no  simple  relationship  between  the  concentration  of  an  acid  in 
which  cohesiveness  is  retained  just  sufficiently  to  hold  the  drop  to 
the  rod  and  the  concentration  of  hydrogen  ions  in  these  soluti6ns 
as  indicated  by  their  conductivities,  although  higher  concentra- 
tions of  weak  than  of  strong  acids  are  as  a  rule  required.  Salts 
lessen  the  power  which  acids  and  alkalies  possess  of  weakening  the 
cohesion  of  gluten,  and  the  concentration  of  salt  required  to  nullify 
the  dispersive  action  of  the  various  acids  varies  with  the  concen- 
tration of  acid  in  a  very  characteristic  manner  which  is  illus- 
trated by  numerous  curves  in  Wood  and  Hardy's  paper.  These 
authors  interpret  their  results  on  the  supposition  that  diminu- 
tion in  the  cohesiveness  of  the  gluten  is  brought  about  by  the 
appearance  of  electric  double  layers  upon  the  surfaces  of  the 
gluten  particles. 

3.  The  Elasticity  of  Protein  Solutions.  —  The  elasticity  of 
protein  solutions  and  jellies  has  been  investigated  by  Rankine 
(89),  Rohloff  and  Shin  jo  (98),  Reiger  (94),  Leick  (58)  and  Hay- 
craft  (42).  They  find  that  even  dilute  solutions  of  gelatin  resist 
deformation  to  a  slight  extent,  a  fact  which  would  appear  to  point 
to  the  existence  within  these  solutions  of  some  species  of  molecular 
(or  ionic)  structure  such  as  that  indicated  in  section  1.  This 
"  form-elasticity "  of  gelatin  solutions  is  diminished  by  heating. 

The  form-elasticity  of  a  freshly  prepared  gelatin  jelly  increases 
with  time  until  it  attains  a  maximum.  According  to  Hay  craft, 
gelatin  jellies  obey  Hooke's  law,  that  is  to  say,  for  moderate 
(linear)  strains  the  deformation  which  is  produced  is  directly  pro- 
portional to  the  applied  force,  and  the  recovery,  when  the  strain 
is  removed,  is  rapid  and  complete. 

According  to  Henderson  and  Brink  (43)  the  compressibility  of 
gelatin  solutions  is  somewhat  less  than  the  compressibility  of  water 
and  lower  the  greater  the  concentration  of  gelatin  in  the  solution. 
With  varying  compression  the  compressibility  varies  in  much  the 
same  way  as  the  compressibilities  of  other  solutions. 


330 


PHYSICAL  PROPERTIES 


4.  The  Diffusion  of  Proteins  in  Solution.  —  It  was  pointed  out 
by  Graham  in  his  classic  memoirs  on  diffusion,  that  the  proteins, 
like  other  colloids,  are  very  sparingly  diffusible.  Nevertheless 
they  diffuse  through  water,  albeit  with  the  extreme  slowness 
indicated  by  their  relatively  enormous  molecular  weight.  From 
the  law  of  Fick  (10)  (30)  we  have: 


where  ds  is  the  quantity  of  diffusing  substance  which  passes  in  the 
time  dz  through  a  diffusion  cylinder  having  the  cross-section  q, 
c  is  the  concentration  of  the  substance  in  the  whole  cross-section 
at  the  point  x,  c  +  dc  is  the  same  quantity  at  the  point  x  +  dx 
and  D  is  a  constant  peculiar  to  the  substance,  expressive  of  its 
diffusibility,  and  known  as  the  "diffusion  coefficient." 

The  following  are  the  diffusion  coefficients  of  certain  proteins, 
estimated  by  the  observers  named  and  cited  after  Wo.  Ostwald 
(81)  (45)  (46).  For  the  purpose  of  comparison  the  value  of  D 
for  sodium  chloride  is  placed  at  the  head  of  the  list. 


Substance 

Observer 

D 

Temperature, 
degrees 

Sodium  chloride 

Voigtlander 

1  040 

20 

Clupein  sulphate 

Herzog 

0  074 

18 

Egg-albumin 

Herzog 

0  059 

18 

Ovomucoid 

Herzog 

0.044 

18 

Albumin 

Graham-Stefan 

0  063 

13 

Now  it  has  been  shown  by  Nernst  (73)  and  Planck  (82)  that  the 
driving  force  which  produces  diffusion  is  osmotic  pressure.  Hence 
we  can  conclude  that  the  proteins,  in  solution,  exert  a  definite, 
although  small  osmotic  pressure. 

From  the  diffusion  coefficient  of  egg-albumin,  Herzog  and 
Kasarnovsky  (46)  have  calculated  that  it  exerts  an  osmotic 
pressure  corresponding  to  a  molecular  weight  of  about  20,000. 
That  proteins  in  solution  exert  a  definite  osmotic  pressure  is  also 
shown  by  cryoscopic,  ebullition,  and  direct  measurements,  of 
which  a  brief  description  will  be  found  below. 

It  has  been  found  by  Dabrovsky  (23)  that  the  diffusion-coeffi- 
cient of  crystallized  egg-albumin  is  decidedly  greater  when  the 
diffusion  takes  place  into  a  solution  of  ammonium  sulphate  than 


FREEZING-  AND  BOILING-POINTS  331 

when  it  takes  place  into  distilled  water.  From  the  value  of  the 
coefficient  of  diffusion  and  the  viscosity  of  the  liquid  into  which 
diffusion  occurs,  it  is  possible,  employing  the  formula  of  Einstein, 
to  compute  the  relative  volumes  occupied  by  the  particles  of  egg- 
albumin  when  diffusing  into  different  solvents.  Applying  this 
method  of  computation,  Dabrovsky  finds  that  the  volume  occu- 
pied by  the  egg-albumin  molecules  when  dissolved  in  distilled 
water  is  no  less  than  six  times  as  great  as  that  which  they  occupy 
when  dissolved  in  a  3.6  per  cent  solution  of  ammonium  sulphate. 
The  extraordinary  diminution  of  molecular  volume  which  am- 
monium sulphate  brings  about  must  undoubtedly  be  connected 
with  the  high  coagulating  power  of  this  salt  and  is  probably  to  be 
attributed  to  dehydration  of  the  protein,  i.e.,  to  abstraction  of  a 
galaxy  of  associated  water-molecules  (Cf.  Chap.  VI,  section  6, 
also  this  Chapter,  section  6). 

5.  The  Freezing-  and  Boiling-points  of  Protein  Solutions.  — 
As  is  well  known,  the  depression  of  the  freezing-point  of  a  solvent 
which  is  produced  by  a  dissolved  substance  is  proportional  to  the 
osmotic  pressure  which  the  dissolved  substance  exerts  in  the  solu- 
tion. Similarly,  the  elevation  of  the  boiling-point  of  the  solvent 
is  proportional  to  the  osmotic  pressure  of  the  dissolved  substance. 
Corresponding  to  the  extremely  high  molecular  weights  of  proteins 
the  osmotic  pressures  of  their  solutions  (containing  practical  per- 
centages of  protein)  are  low,  and  the  depression  of  the  freezing- 
point  and  elevation  of  the  boiling-point  of  water  which  are  brought 
about  by  the  introduction  of  protein  are  small. 

Consequently  Sabanejev  (112)  found  that  the  depression  of 
the  freezing-point  which  is  brought  about  by  egg-albumin  could 
be  almost  entirely  accounted  for  by  the  (estimated)  pressure  of 
the  inorganic  constituents  of  the  protein,  and  Sebanejev  and 
Alexandrov  (114)  estimated  from  their  determinations  that  the 
molecular  weight  of  albumin  must  be  at  least  14,000  in  order  to 
account  for  the  extremely  slight  lowering  of  the  freezing-point 
which  could  be  attributed  to  the  protein  alone.  The  proteoses, 
Sebanejev  estimated,  also  from  cryoscopic  determinations,  to 
possess  a  molecular  weight  of  from  2000  to  3000,  while  the  molec- 
ular weight  of  the  peptones  was  estimated  to  be  400  or  less  (113). 

Tamman  (120)  measured  the  difference  between  the  lowering  of 
the  freezing-point  of  the  serum  of  the  horse  before  and  after  coagu- 
lating the  proteins  by  heat,  and  removing  them,  and  found  that 


332  PHYSICAL  PROPERTIES 

the  difference  amounted  to  only  0.006  degrees,  the  experimental 
error  of  the  cryoscopic  method  being  about  0.005  degrees.  In 
connection  with  experiments  such  as  these  it  should  be  recollected, 
however,  that  heating  induces  many  modifications  in  protein- 
containing  solutions  which  might  conceivably  affect  the  osmotic 
pressure  (freezing-point)  in  a  variety  of  diverse  ways  (92) .  Liide- 
king  found  that  even  40  per  cent  of  gelatin  in  solution  did  not 
perceptibly  alter  the  boiling-point  (67)  and  Krafft  and  Wiglow 
(55)  confirmed  his  results.  Bugarszky  and  Liebermann  (17) 
estimated  the  depression  of  the  freezing-point  due  to  dissolved 
egg-albumin,  albumose  and  pepsin,  and  their  ash,  separately,  and 
deducted  the  latter  from  the  former.  They  estimated  in  this 
manner  the  molecular  (or,  in  reality  mean  molecular  and  ionic) 
weight  of  egg-albumin  to  be  6400,  that  of  albumose  to  be  2400  and 
that  of  pepsin  to  be  760.  They  also  found  that  if  egg-albumin, 
albumose  or  pepsin  be  added  to  solutions  of  acids  or  alkalies,  the 
cryoscopic  depression  of  the  resultant  solution  is  less  than  the  sum 
of  the  depressions  due  to  the  acid  or  alkali  and  the  proteins  dis- 
solved separately;  while  if  the  egg-albumin,  albumose  or  pepsin 
were  dissolved  in  salt  solutions  the  cryoscopic  depression  of  the 
mixture  was  found  to  be  (within  the  limits  of  experimental  error) 
identical  with  the  sum  of  the  cryoscopic  depressions  due  to  the 
protein  and  the  salt  dissolved  separately.  The  significance  of 
these  latter  results  has  already  been  commented  upon  in  Chap.  IV. 
Bugarszky  and  Tangl  (18)  carried  out  an  extended  series  of 
investigations  aiming  at  the  determination  of  the  cryoscopic 
depression  due  to  the  non-electrolytes  of  the  blood,  among  which 
they  included  the  proteins.  They  determined  the  chlorine  con- 
tent of  the  blood  and,  from  that,  deduced  the  equivalent  molec- 
ular concentration  of  the  sodium  chloride  in  the  blood  and  its 
conductivity;  they  then  measured  the  conductivity  of  the  blood 
and  subtracting  from  it  that  due  to  sodium  chloride,  considered 
the  remainder  as  due  to  sodium  carbonate  and  estimated  there- 
from the  molecular  concentration  of  the  sodium  carbonate.  They 
then  subtracted  the  cryoscopic  depression  due  to  the  sodium 
chloride  and  sodium  carbonate  contents  thus  estimated,  from  the 
observed  cryoscopic  depression  of  the  blood;  the  difference  they 
ascribed  to  the  non-electrolytes  and  proteins.  In  this  way  they 
estimated  the  concentration  of  non-electrolytes  in  horse's  blood 
to  be  about  0.056  mol.  per  litre.  The  accuracy  of  this  determina- 


FREEZING-  AND  BOILING-POINTS 


333 


tion  is,  however,  very  questionable,  since  it  depends  upon  the 
assumption  that  the  proteins  in  blood  are  non-electrolytes.  While 
it  is  probable  as  Hardy's  results  indicate  (Cf.  Chap.  VII,  section  6), 
that  the  proteins  in  normal  blood  are  non-ionic,  the  possibility 
must  not  be  lost  sight  of  that  they  may  participate,  as  isolated 
water  soluble  proteins  frequently  do,  in  the  conduction  of  a  cur- 
rent through  their  solutions  (Cf.  Chaps.  VIII  and  X). 

The  majority  of  the  above  estimates  were  not  carried  out  upon 
pure  proteins.  Robertson  and  Burnett  (106),  however,  have 
investigated  the  cryoscopic  depression  due  to  dissolved  casemates 
of  the  alkalies  and  alkaline  earths,  employing  pure  casein.  The 
following  were  the  results  obtained. 

TABLE  I 

Caseinates  containing  50X10~5  equivalents  of  base  per  gram 
(Experimental  error  of  determination  ±0.0025  degrees) 


Base 

Concentration  of 
base  neutralized  by 
casein 

A  =  depression  of 
the  freezing-point 
of  water 

Indicating  a  molec- 
ular plus  ionic 
concentration  of 

NH4OH 

m 

0  045 

m 

NH4OH... 

50 
m 

0  035 

41 
m 

NH4OH 

50 
m 

0  055 

53 

m 

NH4OH... 

33.3 

m 

0  055 

34 

m 

NH4OH  

33.3 
m 

0  07 

34 
m 

NH4OH 

20 
m 

0  095 

26 
m 

KOH    . 

15 
m 

0  0325 

19 

m 

KOH  

50 

m 

0  0375 

57 
m 

KOH 

50 

m 

0  0425 

49 
m 

KOH  

33.3 
m 

0  0475 

44 
m 

KOH  

33.3 

m 

0  05 

39 
m 

KOH    . 

20 
m 

007^ 

37 

m 

20 

25 

334 


PHYSICAL  PROPERTIES 
TABLE  L  — (Continued) 


Base 

Concentration  of 
base  neutralized 
by  casein 

A  =  depression  of 
the  freezing-point 
of  water 

Indicating  a  molec- 
ular plus  ionic 
concentration  of 

KOH 

m 

0  10 

m 

LiOH 

15 
m 

0  03 

18.5 
m 

LiOH  
LiOH 

59.5 
m 
40 

m 

0.045 
0  07 

62 
m 
11 
m 

LiOH  

24 

m 

0  08 

26 

m 

Ca(OH)2 

18 
m 

0  015 

20 

m 

Ca(OH)2  

91 

m 

0  015 

120 
m 

Ca(OH)2  

91 

m 

0  0175 

120 

m 

Ca(OH)2 

91 
m 

0  02 

101 

m 

Ca(OH)2..    .. 

61 

m 

0  02 

92.5 
m 

Ca(OH)2  

61 

m 

0  02 

92.5 
m 

Ca(OH)2  

61 
m 

0.025 

92.5 
m 

Ca(OH)2  

45.5 
m 

0.025 

74 
m 

Ca(OH)2 

45.5 
m 

0  025 

74 
m 

45.5 

74 

FREEZING-  AND  BOILING-POINTS 


335 


TABLE  II 

Caseinates  containing  80X10"5  equivalents  of  base  per  gram 
(Experimental  error  of  determination  ±0.0025  degrees) 


Base 

Concentration  of 
base  neutralized 
by  casein 

A  =  depression  of 
the  freezing-point 
of  water 

Indicating  a  molec- 
ular plus  ionic 
concentration  of 

NH4OH 

m 

0.04 

m 

NH4OH                     

36 

m 

0.04 

46 

m 

NH4OH              

34 

m 

0.0475 

46 
m 

NH4OH  

27 
m 

0.05 

39 

m 

NH4OH 

25 

m 

0.07 

37 
m 

NH4OH 

20 
m 

0.055 

26.5 
m 

NH4OH 

18 
m 

0.075 

34 

m 

NH4OH          .              ... 

17 

m 

0.10 

25 

m 

KOH 

15 

m 

0.035 

18.5 
m 

KOH 

50 

m 

0.04 

53 

m 

KOH                  

33 

m 

0.055 

46 
m 

KOH 

33 

m 

0.06 

34 

m 

KOH 

25 

m 

0.07 

31 

m 

KOH                            

20 

m 

0.0875 

26.5 
m 

KOH                     

17 

m 

0.095 

20 
m 

LiOH     

15 

m 

0.04 

19 

m 

LiOH  

40 
m 

0.05 

46 
m 

LiOH  

30 

m 

0.07 

37 

m 

Ca(OH)2 

20 

m 

0  015 

26 
m 

Ca(OH)2 

91 

m 

0  02 

120 
m 

Ca(OH)2  

68 
m 

0  0225 

92.5 
m 

45.5 

80 

336  PHYSICAL  PROPERTIES 

The  significance  of  these  results  and  of  results  which  I  have 
obtained  with  ovomucoid  is  commented  upon  in  Chap.  X.  It  may 
further  be  remarked  here  that  the  observed  depressions  could  not 
have  been  due  to  impurities  associated  with  the  casein  for  the 
following  reasons: 

(1)  Keeping  the  concentration  of  base  constant  it  is  evident 
that  increasing  the  quantity  of  casein  dissolved  in  it  in  the  pro- 
portion of  8  to  5  does  not  alter  the  observed  depression  in  any 
appreciable  degree,  whereas,  if  this  were  due  to  impurities  associ- 
ated with  the  casein,  it  should  result  in  a  proportionate  increase 
in  the  observed  depression. 

(2)  Keeping  the  concentration  of  casein  constant  and  increasing 
the  concentration  of  base  bound  by  it  in  the  proportion  of  8  to  5 
results  in  a  proportionate  increase  in  the  observed  depression. 

The  cryoscopic  depression  is,  therefore,  obviously  conditioned 
primarily  by  the  combined  alkali.  This  might  be  interpreted  to 
indicate  that  the  depression  is  in  reality  due  to  the  base  and  not  to 
the  protein.  This,  however,  is  not  the  case,  since,  as  we  have  seen 
(Chaps.  V  and  IX),  these  solutions  were  respectively  neutral  to 
litmus  and  to  phenolphthalein  and  therefore  contained  no,  or  only 
a  trace  of,  free  base.  Nor  were  the  depressions  due  to  inorganic 
ions,  since,  as  we  have  seen  (Chap.  VIII),  such  solutions  contain  no 
inorganic  ions.  As  I  have  explained  in  Chap.  X,  the  observed 
depressions  must  be  attributed  primarily  to  protein  ions,  each 
equivalent  of  combined  base  yielding  the  same  number  of  protein 
ions,  derived  through  the  splitting  of  successive  — N.HOC— 
bonds. 

One  fact  should  be  especially  commented  upon  here,  and  that 
is  that  the  observed  depressions  in  the  two  sets  of  solutions  ex- 
amined stood  in  direct  proportion  to  the  concentration  of  combined 
base.  If  this  were  so  for  other  concentrations,  then  at  zero  con- 
centration of  combined  base,  if  casein  were  soluble  under  such 
conditions,  the  freezing-point  depression  due  to  dissolved  casein 
would  be  zero.  In  other  words  the  possibility  is  indicated  that 
base-  and  acid-free  protein  may  exert  an  immeasurably  small 
osmotic  pressure.  I  have  elsewhere  attributed  this  to  polymeri- 
zation of  the  protein  as  the  uncombined  protein  is  set  free  (104) 
(106). 

6.  The  Osmotic  Pressure  of  Proteins  in  Solution.  —  The 
direct  determination  of  the  osmotic  pressure  of  protein  solutions  is 


OSMOTIC  PRESSURE  337 

a  task  fraught  with  immense  difficulties,  on  account  of  the  difficulty 
of  preparing  ideally  pure  proteins.  The  original  investigations 
of  Graham  (35)  appeared  to  indicate  that  colloids  in  general  exert 
a  high  osmotic  pressure.  Subsequent  investigators,  however, 
attributed  these  results  to  an  admixture  of  crystalloids.  Starling 
endeavored  to  measure  directly  the  osmotic  pressure  of  the  pro- 
teins in  blood  serum  by  using  for  his  osmometer  a  membrane 
permeable  to  salts  but  impermeable  to  proteins  (117)  and  this 
method  has  since  then  been  employed  in  all  accurate  work  upon 
the  subject,  since,  as  Reid  has  pointed  out  (92)  it  is  the  only  method 
of  procedure  which  is  applicable  to  the  problem.  We  have  no 
assurance  that  any  given  protein  preparation  is  totally  free  from 
(not  necessarily  conductive)  impurities  which  may  influence  the 
direct  measurement  of  osmotic  pressure;  it  is,  therefore,  essential 
to  employ  a  membrane  which  is  permeable  to  such  impurities  and 
thus,  if  time  be  allowed  for  the  system  to  come  to  equilibrium, 
differentiates  between  protein  and  non-protein  constituents  of  the 
solution  investigated.  For  this  purpose  Reid  employs  a  mem- 
brane of  vegetable  parchment,  which,  as  he  has  shown,  is  per- 
meable even  to  nucleic  acid  although  it  is  impermeable  to  the 
proteins  employed  by  him  in  his  investigations.  By  extremely 
prolonged  purification  Reid  has  succeeded  in  obtaining  prepara- 
tions of  egg-albumin  which  exhibit  no  measurable  osmotic  pressure 
when  examined  by  this  method.  In  subsequent  investigations 
(93),  however,  he  obtained  osmotic  pressures,  due  to  dissolved 
haemoglobin,  of  perfectly  constant  value  and  such  as  to  indicate 
a  molecular  weight  of  about  48,000.  Barcroft  and  Hill  (4)  have, 
however,  demonstrated  by  thermodynamical  methods  that  in 
solutions  containing  haemoglobin  prepared  by  less  prolonged 
dialysis  the  molecular  weight  of  this  substance  is  about  16,669. 
Roaf  (96)  employing  the  differential  osmotic  method  just  de- 
scribed, finds  that  the  molecular  weight  of  haemoglobin,  dissolved 
in  distilled  water,  is  about  32,000,  while  in  sodium  carbonate 
solutions  it  is  16,000.  These  results  appear  to  confirm  the  view 
(Cf.  also  Barcroft  and  Hill  loc.  cit.)  that  non-ionic  protein  is 
polymerized  and  so  exerts  a  considerably  smaller  osmotic  pressure 
than  ionic  protein. 

Benj.  Moore  and  Roaf  and  collaborators  (70)  (71)  (72)  (95)  (97) 
and  R.  S.  Lillie  (63)  have  made  the  extremely  interesting  discovery 
that  the  osmotic  pressure  which  is  exerted  by  proteins  (deter- 


338 


PHYSICAL  PROPERTIES 


mined  differentially,  as  described)  varies  very  pronouncedly  with 
the  nature  of  the  inorganic  acids,  bases  or  salts  which  their  solu- 
tions contain.* 

The  following  are  among  Lillie's  results,  obtained  when  dilute 
acids  and  alkalies  were  employed  as  solvents: 

TABLE  III 
1.5  Per  Cent  Gelatin  in  Dilute  HC1  Solutions 


Solvent 

Osmotic  pressure  of 
protein  in  mm.  Hg. 

Water  
ra/3100  HC1  
m/2050  HC1  
m/1550HCl.-  
ra/1025HCl  
ra/770  HC1  

8.2 
6.8 
12.3 
17.9 
26.5 
32.4 

ra/620  HC1  
ra/412  HC1 

34.9 
39.3 

TABLE  IV 
1.5  Per  Cent  Gelatin  in  Dilute  KOH  Solutions 


Solvent 

Osmotic  pressure  of 
protein  in  mm.  Hg. 

Water 

7.9 

w/3100  KOH 

14.1 

m/620  KOH  

23.7 

w/412  KOH 

25.1 

m/310  KOH 

29.0 

In  Lillie's  words,  "In  the  presence  of  either  acid  or  alkali  the 
osmotic  pressure  of  gelatin  thus  shows  a  marked  increase,  which, 
within  the  above  range  of  concentrations,  exhibits  a  certain  pro- 
portionality to  the  quantity  of  acid  or  alkali  added.  For  equiva- 
lent concentrations,  acid  produces  a  somewhat  greater  increase 
than  alkali.  The  change  in  osmotic  properties  is  to  be  attributed 
to  a  finer  subdivision  of  the  colloid  particles  and  a  consequent 
increase  in  the  surface  of  intersection  between  colloid  particles  and 

*  In  this  connection  it  is  of  interest  to  note  that  von  Wittich  (131),  von 
Regeczy  (91),  Oker-Blom  (76)  and  others  have  shown  that  proteins  diffuse 
mote  rapidly  into  sodium  chloride  solutions  than  they  do  into  distilled  water. 
Cf.  also  Dabrovsky  (23). 


OSMOTIC  PRESSURE  339 

medium."  These  results  are  obviously  in  complete  agreement 
with  results  obtained  by  Robertson  and  Burnett,  employing  the 
cryoscopic  method,  which  are  commented  on  above.  The  decrease 
in  pressure  observed  in  very  dilute  acid  may  be  attributable  to  the 
neutralization  by  the  acid  of  base  bound  by  the  protein  prepara- 
tion employed.  Lillie  draws  an  analogy  between  his  results  and 
those  obtained  by  Ostwald  (78)  (79)  in  investigating  the  swelling 
of  gelatin  plates  in  dilute  acid  and  bases.  Egg-albumin,  how- 
ever, exhibits  the  opposite  behavior,  namely,  the  osmotic  pressure 
which  it  exerts  is  slightly  diminished  by  acids  and  bases. 

The  osmotic  pressure  of  gelatin  and  egg-albumin  is  unaffected 
by  the  addition  of  non-electrolytes  such  as  cane-sugar,  dextrose, 
glycerol  and  urea,  but  is  considerably  affected  by  the  addition  of 
inorganic  salts  being  (and  this  is  true  both  for  gelatin  and  egg- 
albumin)  depressed  thereby.  According  to  Lillie  the  depression 
of  the  osmotic  pressure  exerted  by  this  protein  is  a  function  of  the 
nature  of  both  the  anion  and  cation  of  the  added  salt.  It  increases 
in  the  order  alkali  metals  <  alkaline  earths  <  heavy  metals  (for 
cations) ;  and  CNS  <  I  <  Br  <  N03  <  Cl  <  F  <  plurivalent 
anions,  S04,  tartrate,  citrate,  phosphate  (for  anions).  This 
observation  is  of  extraordinary  significance  when  we  recollect  that 
this  is  the  order  in  which,  according  to  Hofmeister  and  Pauli,  the 
various  ions  bring  about  the  dehydration  and  coagulation  of  the 
salts  which  proteins  form  with  bases  (Cf.  Chap.  VI).* 

Very  remarkable  phenomena  are  displayed  by  the  solutions  of 
soluble  chitin  which  have  been  prepared  by  Alsberg  and  Hedblom 
(1)  from  the  chitin  of  Limulus  polyphemus  by  prolonged  treatment 
with  weak  hydrochloric  acid.  The  chitin  when  subjected  to  this 
treatment  forms  at  first  a  gelatinous  mass  and  later  a  colloidal 
solution.  The  analytical  figures  obtained  for  this  "soluble 
chitin"  and  the  gelatinized  chitin  obtained  by  adding  KOH  and 
then  reneutralizing  with  HC1  are  best  explained,  according  to  the 
authors,  by  assuming  that  the  chitin,  on  passing  into  colloidal 
solution,  unites  with  water.  Soluble  chitin  depresses  the  freezing 
point  but  slightly,  so  that  its  molecular  weight  is  probably  very 
high.  It  passes  through  collodion  and  parchment  paper,  but  has 
the  extraordinary  property  of  carrying  the  water  in  which  it  is 

*  Moore  and  Roaf  (72)  have  sought  to  explain  the  coagulation  of  proteins 
by  salts  by  the  formation  of  large  aggregates,  basing  their  argument  upon  data 
similar  to  these  obtained  by  Lillie. 


340  PHYSICAL  PROPERTIES 

dissolved  through  the  membrane  so  that  the  space  within  the 
latter  may  become  nearly  empty.  It  is  probable  that  these  phe- 
nomena depend  rather  upon  the  extreme  affinity  of  this  colloid  for 
water  than  upon  ordinary  osmotic  forces. 

C.  J.  Martin  (68)  and  W.  M.  Bayliss  (5)  find  that  the  os- 
motic pressures  of  egg-albumin  and  of  haemoglobin  solutions  vary 
directly  with  the  absolute  temperature,  thus  obeying  the  law  of 
Gay-Lussac. 

7.  The  Nature  of  Protein  Solutions.  —  The  view  has  been  held 
by  many  investigators  that  the  proteins,  and  colloids  generally,  do 
not  enter  into  true  solution,  forming  molecularly  dispersed  systems 
which  obey  the  law  of  Avogadro,  but  are  to  be  regarded,  on  the 
contrary,  as  forming  stable  suspensions  when  they  appear  to  pass 
into  solution  in  water;  a  view  which  reaches  its  most  extreme 
expression  in  the  statement  of  Duclaux  (26)  that  "Les  Colloids 
doivent  etre  consideres  comme  etant,  dans  Peau,  d'une  insolubilite 
absolue."  Others,  arguing*  from  the  a  priori  assumption  that 
"solutions"  of  colloids  are  necessarily,  in  reality,  suspensions,  have 
objected  to  the  application  of  theoretical  principles  to  them  which 
involve  the  law  of  Avogadro  (such  as  the  mass-law,  etc.).  It  is 
obvious  that  this  is  a  reversion  of  the  customary  procedure  of 
science;  the  applicability  of  a  law  is  in  the  first  place  a  question  in 
itself,  independent  of  any  other  generalizations,  and,  in  the  second 
place,  the  applicability  of  a  law  involving  Avogadro's  law  may  in 
itself  be  regarded  as  presumptive  evidence  that  the  law  of  Avo- 
gadro applies  as  well. 

From  what  has  been  said  above,  it  is  evident  that  under  certain 
conditions,  particularly  in  the  form  of  their  salts,  the  proteins 
diffuse  in  water  and  exert  a  definite  osmotic  pressure.  Hence 
Avogadro's  rule  must  hold  good,  although  the  time  required  for 
the  protein  molecules  to  distribute  themselves  uniformly  through- 
out any  given  volume  of  fluid  may  conceivably,  in  certain  instances, 
be  very  great. 

Apart  altogether  from  direct  observation,  however,  the  posses- 
sion of  a  definite  osmotic  pressure  by  proteins  and  certain  other 
colloids  in  solutions  is  directly  deducible  from  the  fact  that  chemi- 
cal reactions  which  involve  them  attain  definite  equilibria  (101). 
The  work  which  is  performed  in  the  transformation  of  a  molecule 
of  substance  is  the  sum  of  two  factors,  the  one  the  chemical  work 
*  For  example,  W.  Nernst  (75). 


NATURE  OF  PROTEIN  SOLUTIONS         341 

which  is  involved  in  the  transformation,  the  other  the  work  per- 
formed in  bringing  the  molecule  to  the  pressure  of  the  system  (56) ; 
the  latter  factor  is,  of  course,  dependent  upon  the  concentration  of 
the  substances  within  the  system,  while  the  former,  equally 
obviously,  is  not.  If  the  reacting  components  in  a  system  exerted 
no  osmotic  pressure  whatever,  the  expression  for  the  work  done  in 
the  transformation  of  a  given  mass  of  the  components  would, 
therefore,  be  a  constant  one  (depending  only  upon  temperature), 
and,  consequently,  at  any  stage  of  the  reaction  the  work  done  in 
transforming  unit  mass  would  be  the  same,  whatever  the  concen- 
tration of  the  reacting  components.  Under  these  conditions  the 
reaction  would  always  proceed  to  an  end  in  one  direction  or  the 
opposite;  since  the  work  performed  at  every  stage  of  the  reaction 
would  be  unaffected  by  the  concentration  of  the  reacting  com- 
ponents. Now  it  has  been  shown  that  the  reactions  between 
toxins  and  anti-toxins,  lysins,  and  anti-lysins,  etc.,  attain  definite 
equilibria  (3)  and  hence  these  bodies  must,  in  solution,  exert 
definite  osmotic  pressure.  The  probable  protein  nature  of  these 
bodies  has  been  commented  upon  in  a  previous  chapter  (Chap. 
VII,  section  7).  Moreover,  it  has  been  shown  that  a  definite 
equilibrium  is  attained  between  proteins  and  the  antibodies  which 
are  produced  in  the  serum  when  these  proteins  are  injected  into 
the  circulation  (3),  and  definite  equilibria  are  attained  between 
different  proteins  in  solution  (121)  and  between  proteins  and  in- 
organic acids  and  bases  (Cf.  Chap.  IX)  and  in  protein  digests  (122) 
(123)  (99)  (102)  (107).  Many  of  these  equilibria,  it  has  been 
shown,  can  be  approached  from  either  direction,  so  that  they  are 
not  " false"  equilibria  (27)  attributable  to  the  internal  molecular 
friction  or  hysteresis  of  the  systems.  We  may,  therefore,  safely 
conclude  that  in  many  instances,  and  especially  in  the  form  of 
their  salts,  proteins  and  bodies  allied  to  proteins  exert,  when  in 
solution,  definite  osmotic  pressures;  and  are  distributed  in  molec- 
ular dispersion  throughout  their  solutions  in  accordance  with  the 
law  of  Avogadro. 

It  is,  of  course,  not  for  a  moment  contended  that  this  is  the  case 
in  every  apparent  "solution"  of  protein.  Proteins,  like  other 
bodies,  and  more  easily  than  the  majority  of  crystalloids,  can  also 
be  obtained  in  a  suspended  condition.  Under  such  circumstances 
they  may  form  apparently  stable  suspensions,  simulating  true 
solutions  in  certain  respects.  An  illustration  of  such  suspension 


342  PHYSICAL  PROPERTIES 

is  afforded  by  potassium  caseinate  in  75  per  cent  alcohol  (Cf. 
Chap.  XI). 

Certain  authors,  following  Graham,  regard  all  colloidal  solu- 
tions, including  protein  solutions,  as  being  "  supersaturated." 
The  above  considerations  dispose  of  this  view  also.  Linebarger 
(64)  has  also  shown,  by  direct  experimentation  on  the  rate  of  coagu- 
lation, that  solutions  of  proteins  cannot  be  correctly  regarded  as 
supersaturated.  Very  numerous  observations,  detailed  in  previous 
chapters,  will  occur  to  the  reader  which  also  conflict  with  this  view, 
for  example,  the  constancy  of  the  proportion  between  base  and 
casein  at  " saturation"  of  the  former  with  the  latter. 

8.  The  Opalescence  of  Protein  Solutions;  the  Tyndall  Effect. 
—  The  majority  of  protein  solutions  are  decidedly  opalescent, 
i.e.,  they  contain  particles  of  sufficient  size  to  scatter  and  reflect  a 
proportion  of  transmitted  light.  Even  when  the  opalescence  is 
very  small,  when  the  solution  is  viewed  at  right  angles  to  the 
direction  of  a  pencil  of  light  traversing  it,  a  certain  degree  of 
scattering  (the  Tyndall  effect)  is  observed.  Upon  this  fact  many 
investigators  have  based  the  view  that  proteins  in  solution  form 
heterogeneous  and  not  molecularly  dispersed  systems. 

It  has,  however,  been  pointed  out  by  Konovalov  (53)  that  the 
dust  particles,  which  render  it  so  difficult  to  obtain  even  the  purest 
water  in  an  "optically  void"  condition,  may  act  as  nuclei,  under 
conditions  in  which  the  expenditure  of  energy  necessary  for  an 
alteration  in  concentration  is  very  small  (i.e.,  when  the  osmotic 
pressure  of  the  dissolved  substance  is  very  small),  attracting 
layers  of  the  dissolved  substance  and  so  producing  opalescence. 
In  this  way  it  is  possible  that  the  observed  opalescence  in  such 
solutions  is  due  only  to  a  minute  proportion  of  the  dissolved  sub- 
stance condensed  by  capillary  action  around  accidental  insoluble 
contaminations.  Arrhenius  (3)  has  advanced  a  similar  view.  "It 
may,  however,  be  urged  that  the  presence  of  some  submicroscopic 
particles  does  not  prove  at  all  that  the  whole  of  the  quantity  of 
protein  present,  or  even  a  considerable  part  of  it,  is  in  this  state 
of  pseudo-solution." 

It  is,  however,  not  at  all  inconceivable  that  the  opalescence  of 

many  colloidal  solutions  may  be  due  to  the  grossness  of  the  colloid 

molecules  themselves,*  but  it  is  unlikely  that  this  is  the  case  in 

solutions  of  proteins.     It  has  been  shown  by  Lobry  de  Bruyn  (16) 

*  C.  O.  Weber  (128),  p.  75. 


OPALESCENCE  343 

that  the  smallest  particles  which  are  capable  of  scattering  light  are 
from  fifty  to  a  hundred  times  smaller  than  the  mean  wave  length 
of  light  at  0.5  /*,  the  diameter  of  such  particles  could  be  not  less 
than  from  5  to  10  MM-  Now  we  have  seen  in  Chaps.  VIII  and  IX, 
that  the  molecular  weight  of  casein  in  solutions  containing  11.4  X 
10~5  equivalents  of  base  per  gram  of  protein,  is  probably  about 
17,600  (estimated  from  electrochemical  data).  From  the  analysis 
of  Hammarsten  (39)  it  appears  that  if  casein  be  possessed  of  this 
molecular  weight,  then  its  empirical  formula  must  be,  approxi- 
mately: 


Now  it  has  been  shown  by  Kopp  (54)  that  the  relative  molecular 
volumes  of  organic  substances  can,  with  certain  marked  exceptions, 
be  calculated  as  an  additive  function  of  their  constituent  atoms.* 
The  mode  in  which  the  valencies  of  the  atoms  are  distributed 
affects  their  contribution  to  the  total  volume,  however,  "carbonyl" 
oxygen  contributing  an  amount  differing  from  that  contributed  by 
hydroxyl  oxygen.  From  the  analysis  of  Abderhalden  and  others  it 
would  appear  that  if  casein  possesses  the  above  molecular  weight 
it  must  contain  about  four  molecules  of  tyrosin,  and  one  each  of  the 
other  hydroxy-acids  which  it  is  known  to  contain,  namely,  serin 
and  caseinic  acid,  or  in  all,  neglecting  the  necessarily  few  terminal 
—  COOH  groups  (Cf.  Chap.  I)  it  would  appear  to  contain  only 
about  8  atoms  of  hydroxyl  oxygen  out  of  the  250  atoms  of  oxygen 
which  are  present.  But  it  will  be  recollected  (Chap.  I)  that  there 
is  strong  reason  to  suspect  that  in  many  of  the  —  COHN  —  groups 
within  the  protein  molecule  the  oxygen  is  in  the  hydroxy  condition. 
We  must,  therefore,  make  two  estimates  of  the  molecular  volume, 
one  on  the  supposition  that  the  molecule  contains  only  carbonyl 
oxygen  and  the  other  on  the  supposition  that  it  contains  only 
hydroxyl  oxygen.  The  true  volume  will,  it  is  probable,  lie  be- 
tween the  two.  Now  the  difference  in  molecular  volume  due  to 
carbon  is  11,  that  due  to  hydrogen  is  5.5,  to  nitrogen  (as  in  am- 
monia) 2.3,  to  carbonyl  oxygen  12.2,  to  sulphur  22.6,  to  phos- 
phorus 25.5,  yielding,  for  the  first  estimate  of  the  relative  molecular 
volume  of  casein: 

777  X  11  +  1241  X  5.5  +  197  X  2.3  +  250  X  12.2  +  4  X  22.6 
+  5  X  25.5  =  19,060; 

*  The  figures  employed  in  the  calculation  which  follows  are  cited  after 
Ostwald  (77). 


344  PHYSICAL  PROPERTIES 

a  second  estimate,  regarding  the  oxygen  as  hydroxyl  oxygen 
(difference  due  to  one  atom  of  hydroxyl  oxygen  =  7.8),  yields  the 
molecular  volume  17,960.  The  volume,  estimated  in  this  way, 
therefore,  lies  between  18,000  and  19,000.  The  absolute  volume  of 

i       i       -n  +u     't       r    u  18,000       ,  19,000        , 

the  molecule  will  therefore  he  between  — - —  and  —        •  or  about 

o.o  o.o 

3400  times  that  of  a  hydrogen  molecule,  and  about  523  times  that 
of  a  molecule  of  carbon  dioxide.  The  diameter  of  the  molecule 
will,  therefore,  be  approximately  v^523  =  about  8  times  that  of  a 
molecule  of  carbon  dioxide.  Now  the  diameter  of  a  molecule  of 
C02  is,  according  to  Nernst  (74),  0.3  w,  hence  that  of  a  molecule 
of  casein  must  be  about  2.4  /*/*,  or  about  one-half  the  diameter  of 
the  smallest  particle  which  will  scatter  transmitted  light.  Ad- 
mitting the  great  uncertainty  which,  in  our  present  state  of 
incomplete  knowledge,  attaches  to  such  estimations  as  these,  we 
must  yet  admit  that  it  is  improbable  that  the  opalescence  of  solu- 
tions of  proteins  such  as  casein  is  directly  attributable  to  the 
protein  molecules  themselves;  and  it  appears  the  more  improbable 
when  we  recollect  that  in  pronouncedly  opalescent  solutions  casein 
salts,  and  many  other  protein  salts  are,  as  we  have  seen  (Chaps. 
VIII-X),  quite  extensively  ionized,  that  is,  split  up  into  particles 
which  occupy,  themselves,  a  fraction  of  the  volume  computed 
above.  When  we  recollect  that  the  non-filterable  character  of 
many  proteins  in  solution  (Cf.  Chap.  VII,  section  6)  and  the  vis- 
cosity of  protein  solutions  (Cf .  the  earlier  part  of  this  chapter)  are 
alike  attributable  to  ionic  protein,  the  suggestion  offers  itself  that 
the  opalescence  of  protein  solutions  (as  distinguished  from  that  of 
suspensions  of  partially  coagulated  protein)  may  possibly  be 
attributable  to  the  peculiar  characteristics  of  ionic  protein.  It  is 
possible  that  the  non-filterable  character  of  ionic  protein  and  its 
power  of  scattering  transmitted  light  are  alike  due  to  a  bulky 
water-complex,  which  is  associated  with  each  of  the  protein  ions 
(Cf.  Chap.  VI,  section  6  and  Chap.  VII,  section  6).  In  support  of 
this  view  may  be  mentioned  the  remarkable  fact  that  the  addition 
of  alcohol  or  of  acetone,  in  quantities  insufficient  to  initiate  coagu- 
lation, to  solutions  of  the  caseinates  leads  to  a  very  decided  de- 
crease in  their  opalescence.  On  the  other  hand,  it  is  possible  that 
the  net-like  structure  which  we  have  seen  reason  to  believe  that 
protein  ions  form  in  solution  (section  1,  this  chapter)  is  account- 
able, not  only  for  the  viscosity  of  solutions  of  ionic  protein,  but 


SURFACE  TENSION  345 

also  for  the  non-filterability  of  ionic  protein  and  the  opalescence 
of  its  solutions.  Between  these  alternatives,  if  indeed  they  are 
alternatives,  our  data  do  not,  as  yet,  suffice  for  us  to  decide. 

The  opalescence  of  protein  suspensions  has  been  made  by  Kober 
(52)  the  basis  of  a  method  of  determining  proteins  quantitatively 
(Cf.  Chap.  III). 

9.  The  Surface  Tension  of  Protein  Solutions.  —  The  influence 
of  dissolved  protein  upon  the  tension  of  air-water  surfaces  has  been 
investigated  by  Quincke  (85)  (86),  Zlobicki  (134),  Frei  (31),  Buglia 
(19),  Iscovesco  (59),  Shorter  (115),  Bottazzi  (13)  (15),  and  Berc- 
zeller  (9).     Gelatin,  egg-globulin  and  haemoglobin  diminish  the 
air- water  tension.*    The  diminution  is  greater  the  higher  the 
temperature  (between  0  and  24.4  degrees,  Zlobicki).     The  surface- 
tension  of  gelatin  solutions  and  blood  serum  is  increased  by  the 
addition  of  small  quantities  of  alkali,  and  diminished  by  the 

'addition  of  small  quantities  of  acid  (Buglia,  Frei). 

According  to  Bottazzi  (13)  (15)  the  surface  tension  of  serum 
albumin  solutions  is  at  a  maximum  when  their  ionization  is  at  a 
minimum  and  at  a  minimum  when  their  ionization  is  at  a  maxi- 
mum. The  maximum  depression  of  the  surface  tension  of  water 
by  serum  albumin  occurs  at  a  reaction  just  on  the  acid  side  of 
neutrality.  Undissolved  protein  does  not  affect  the  surface  ten- 
sion of  water  when  shaken  up  with  it  (9). 

According  to  Berczeller  (9),  the  surface  tension  of  a  protein 
solution  which  is  so  electrolyte-free  as  not  to  coagulate  on  heating 
nevertheless  diminishes  on  heating.  This  reduction  of  surface 
tension  is  reversible,  for  on  standing  for  some  time  at  ordinary 
temperature  the  solution  regains  its  original  surface  tension. 

The  surface  tension  of  protein  solutions  diminishes  during 
digestion  (9). 

10.  The  Formation  of  Surface-films  by  Dissolved  Proteins.  — 
Proteins,  when  dissolved  in  water,  would  appear,  as  a  rule,  to 
diminish  the  surface  tension  at  surfaces  bounded  by  substances 
other  than  air,  since  they  tend  to  become  concentrated  at  such 
surfaces  and  form  films  of  insoluble  protein  there,  and,  as  Willard 
Gibbs  pointed  out  in  his  classic  memoir  on  equilibrium  in  hetero- 
geneous systems,  such  a  tendency  is  indicative  of  a  diminution  in 
the  free  energy  of  the  surface  at  which  such  concentration  occurs. 

*  According  to  Iscovesco,  however,  pure  egg-albumin  raises  the  air-water 
tension. 


346  PHYSICAL  PROPERTIES 

This  phenomenon  has  received  extended  consideration  at  the  hands 
of  Hermann  (44),  Berthold  (11),  Ramsden  (87)  (88),  and  Shorter 
(115),  who  have  shown  that  protein  solutions  can  be  wholly 
or  partially  coagulated  by  mere  mechanical  agitation,  or  by  the 
purely  surface-action  of  fine  powders,  such  as  burnt  clay  or  char- 
coal. Ramsden  also  explains  the  formation  of  a  film  at  the  surface 
of  heated  milk  and  other  protein  solutions  in  the  same  way.* 

The  formation  of  these  protein  films  by  surface-action  can  be 
very  conveniently  illustrated  by  the  following  experiments  (100) 
(103).  If  chloroform  be  shaken  up  with  casein,  gelatin  or  pro- 
tamin  solutions,  it  settles  in  fine  particles  or  droplets,  which,  if 
numerous,  form  a  milky  layer  at  the  bottom  of  the  vessel;  by 
transmitted  light,  however,  they  appear  perfectly  clear.  These 
droplets  are  extraordinarily  stable,  and  do  not  coalesce,  however 
long  they  stand  in  contact;  they  may  be  repeatedly  washed  in 
water  until  all  traces  of  protein  have  been  removed  from  the  super- 
natant fluid,  and  they  still  remain  perfectly  stable  and  distinct 
from  one  another;  they  may  be  shaken  up  in  chloroform  itself  or 
treated  with  JV/10  potassium  hydroxide  without  impairing  their 
form  or  stability.  If,  however,  they  be  heated  to  nearly  the 

*  The  non-filterability  of  many  proteins,  that  is,  their  inability  to  pass 
through  the  pores  of  a  clay  filter  has,  by  some  authors,  also  been  attributed  to 
surface-action.  This  factor  alone,  however,  is  insufficient  to  explain  the  total 
non-filterability  of  these  proteins  through  certain  filters.  True,  when  solutions 
of  substances  which  reduce  the  surface-tension  at  the  surfaces  of  insoluble 
powders  are  filtered  through  such  powders,  the  first  portion  of  the  filtrate 
which  filters  through  contains  less  of  the  dissolved  substance  than  the  original 
solution  (Cf.  J.  J.  Thomson  (124));  for  example,  when  potassium  perman- 
ganate solution  is  filtered  through  fine  quartz  sand  the  first  portions  of  the 
nitrate  may  be  colorless.  But  subsequent  portions  of  the  nitrate  contain 
a  greater  proportion  of  the  solute  and,  finally,  the  unaltered  solution  filters 
through,  owing  to  the  fact  that  the  surfaces  of  the  pores  of  the  filter  are 
now  fully  coated  by  a  film  of  more  than  molecular  thickness.  If  this  were 
the  only  factor  concerned  in  holding  back  dissolved  proteins  from  passing 
through  clay  filters,  the  protein  would  sooner  or  later  pass  through  the  filter, 
which,  in  many  cases  it  does  not  (Cf.  F.  W.  Zahn  (133),  J.  Lehmann  (57)). 
True,  it  is  possible  that  the  first  portions  of  the  protein  which  are  de- 
posited upon  the  walls  of  the  pores,  partially  clog  up  the  pores  and  narrow 
them,  thus  assisting  the  filter  to  hold  the  protein  back;  but  even  if  this  be  the 
case,  the  ultimate  factor  which  determines  the  impermeability  of  such  filters 
for  proteins  must  either  be  the  grossness  of  the  protein  particles  themselves, 
or  of  their  associated  water-complexes,  or  else  the  existence  within  the  protein 
solution  of  a  structure  connecting  together  the  protein  particles. 


SURFACE-FILMS  347 

boiling  point  of  chloroform,  under  a  layer  of  water,  the  droplets 
burst  and  coalesce,  forming  a  homogeneous  layer  of  chloroform. 
If  treated  with  alcohol  they  immediately  dissolve,  leaving  a  fine 
membraneous  precipitate  of  protein  floating  in  water.  Thus,  if  we 
shake  up  chloroform  with  about  twice  its  volume  of  a  1  per  cent 
solution  of  protamin  sulphate,  and,  after  standing,  separate  the 
chloroform  droplets  by  pouring  off  the  supernatant  liquid,  and 
then,  after  repeatedly  washing  the  droplets  in  water  by  decanta- 
tion,  add  to  the  small  amount  of  supernatant  water  about  an  equal 
volume  of  alcohol  and  gently  agitate,  the  droplets  which  are  thus 
stirred  up  into  the  alcohol-water  layer  can  be  seen  to  swell  up 
rapidly  and  burst,  and  the  fine  membranes  which  surround  them 
can  be  seen  falling  down  through  the  alcohol-water.  If  we  now 
add  several  volumes  of  alcohol  and  shake  up  the  liquid,  the  chloro- 
form droplets  all  disappear,  and  what  we  now  have  is  a  clear, 
homogeneous  solution  in  which  innumerable  minute  membranes 
can  be  clearly  seen  floating.* 

That  these  surface  films  possess  quite  different  properties  from 
the  protein  in  the  body  of  the  fluid  is  evident;  this  is  shown  by 
their  great  insolubility  and  by  the  fact  that  they  are  not  readily 
acted  upon  by  acids  and  alkalies.  Now  many  investigators  have 
pointed  out  that  at  the  surface  separating  two  phases  of  a  system 
marked  changes  in  chemical  equilibrium  frequently  occur  (74) 
(62)  (48),  owing  to  the  alteration  in  chemical  potential  which 
occurs  at  such  surfaces  (124).  It  appears  probable  that  at  surfaces 
within  protein  solutions  similar  changes  in  equilibrium  occur, 
leading  to  the  formation  of  polymers  or  anhydrides  of  the  protein.f 
These  alterations  in  equilibrium  are  very  slowly  reversed  upon 
removal  of  the  protein  from  the  surface  which  caused  the  change 
in  equilibrium;  this  is  shown  by  the  insolubility  of  the  coagula 
which  are  produced  in  this  way.  Evidently  some  phenomenon 
analogous  to  hysteresis  prevents  or  greatly  delays  complete  return 

*  The  formation  of  these  films  also  explains  the  power  which  proteins 
possess  to  render  many  emulsions  stable,  which  are  not  stable  in  pure  water 
(Cf.,  for  example,  Jamison  and  Hertz  (50)).  It  also  explains  the  tendency  of 
protein  solutions  to  form  foams.  For  the  analogous  part  which  is  played  by 
soap  films  Cf.  Quincke  (84)  and  T.  Brailsford  Robertson  (104). 

t  In  this  connection  it  should  be  noted  that  the  increase  in  the  concentra- 
tion of  proteins  at  such  surfaces,  due  to  the  diminution  in  the  surface  energy 
which  the  protein  causes,  must  be  accompanied  by  a  corresponding  diminution 
of  the  active  mass  of  water  at  these  surfaces. 


348  PHYSICAL  PROPERTIES 

to  the  equilibrium  which  was  disturbed.  The  occurrence  of  such 
phenomena  of  hysteresis  in  heterogeneous  systems  containing 
proteins,  and  their  probable  dependence  upon  the  internal  friction 
of  the  solid  phase  have  been  commented  upon  in  connection  with 
the  heat-coagulation  of  proteins  (Cf.  previous  chapter). 

I  have  shown  that  the  films  which  are  formed  by  gelatin  around 
chloroform  droplets  are  permeable  to  alcohol  and  also,  but  less, 
permeable  to  chloroform.  The  membranes  are  permeable  to 
various  substances  in  the  following  order,  those  to  which  they  are 
most  permeable  being  placed  first,  alcohol,  ether,  ethyl  acetate, 
scharlach  R.,  chloroform,  toluol,  xylol,  carbon  bisulphide. 

The  interesting  observation  has  been  made  by  Shorter  (115) 
that  the  surface-elasticity  of  freshly  prepared  protein  solutions 
undergoes  a  progressive  increase  with  time.  These  increases  are 
not  uniform  but  step-like,  and  represent,  with  irregular  fluctua- 
tion, the  deposition  of  successive  layers  of  molecular  thickness. 
Equilibrium  is  only  attained  with  extreme  slowness,  a  phenome- 
non which  Shorter  attributes  to  the  fact  that  in  solutions  an  in- 
crease in  concentration  of  the  protein  at  any  point  has  a  large 
effect  upon  surface-tension  but  only  a  small  effect  upon  the  osmotic 
pressure  of  the  underlying  layers  of  solution.  In  concentrated  so- 
lutions of  protein,  after  a  very  prolonged  period,  it  was  found  in 
some  cases  that  the  progressive  thickening  of  the  surface-membrane 
was  suceeded  by  thinning.  It  is  not  certain,  however,  where  such 
long  periods  of  time  are  concerned,  that  autohydrolysis  of  the  pro- 
tein may  not  have  contributed  toward  modification  of  the  results. 

The  possible  biological  significance  of  protein  films  of  this  char- 
acter has  been  discussed  at  some  length  in  my  communications 
referred  to  above. 

11.  The  Specific  Gravities  of  Protein  Solutions.  —  We  have 
seen  (Chap.  XII)  that  the  taking  up  of  water  by  concentrated 
proteins  (gelatin)  is  accompanied  by  a  volume-contraction  (67); 
hence  in  concentrated  protein  solutions  the  specific  gravity  cannot 
be  calculated  directly  from  the  specific  gravities  of  the  solvent  and 
solute  respectively. 

The  specific  gravities  of  dilute  protein  solutions  have  been  in- 
vestigated by  Haebler  (38),  Lehnstein  (66)  and  others.  According 
to  the  latter  author,  the  change  in  the  specific  gravity  of  a  sodium 
hydroxide  solution  due  to  the  introduction  of  protein  is  directly 
proportional  to  the  percentage  of  protein  introduced. 


SPECIFIC  GRAVITY  349 

Chick  and  Martin  (21)  have  compared  the  densities  of  proteins 
in  the  dissolved  and  in  the  dry  conditions  and  find  invariably  an 
increase  in  density  of  the  dissolved  protein  corresponding  to  the 
volume-contraction  which  accompanies  the  passage  of  proteins 
into  solution.  Thus  dry  casein  suspended  in  benzol  has  a  specific 
gravity  of  1.318,  while  a  7.85  per  cent  solution  of  casein  in  dilute 
sodium  hydroxide  has  a  specific  gravity  of  1.0240,  whence  the 
density  of  the  dissolved  sodium  caseinate  =  1.42,  while  correcting 
for  the  effect  due  to  sodium  hydroxide,  the  dissolved  casein  itself 
has  a  density  of  1.39. 

The  following  table  summarizes  the  results  obtained  with 
various  proteins: 


Protein 

Density  in  solution 

Density  in  the  dry 
state 

Casein  

1.390 

1.318 

Crystallized  egg-albumin  

1.359 

1.269 

Crystallized  serum-albumin  

1.378 

1.275 

Serum  globulin 

1  374 

1  293 

The  more  dilute  a  solution  of  sodium  caseinate,  the  greater  its 
apparent  density  was  found  to  be.  This  was  not  the  case  in  solu- 
tions of  serum  globulin,  or  albumin. 

12.  The  Magnetic  Properties  of  the  Proteins.  —  It  was  shown 
in  1845  by  Faraday  (29)  and  later  by  Pliicker  (83)  that  haemo- 
globin contains  iron;  it  is,  nevertheless,  like  the  other  constituents 
of  living  tissues,   decidedly  diamagnetic.     This  observation  has 
been  confirmed  in  an  extended  series  of  investigations  by  Gamgee 
(32),  who  finds  that  not  only  oxyhsemoglobin  but  also  CO-haemo- 
globin  and  methsemoglobin  are  strongly  diamagnetic  bodies.     The 
iron-containing  radical,  hsematin,  and  its  derivative  hsemin  are, 
on  the  contrary,  as  might  be  expected  from  their  content  of  iron, 
strongly  magnetic  bodies. 

Benedicenti  (7)  (8)  finds  that  the  presence  of  protein  inhibits 
the  lowering  of  the  diamagnetic  constant  of  water  by  the  addition 
of  dissolved  ferric  chloride  or  finely  pulverized  iron. 

13.  "The  Gold-Number"  of  Proteins. —  It  was  found  by 
Zsigmondy  (135)  that  colloidal  gold  is  precipitated  from  its  solution 
(or  suspension)  by  the  addition  of  sodium  chloride,  but  that  this 
precipitation  can  be  prevented  by  the  addition  to  the  solution  of 
many  other  colloids,  among  which  may  be  reckoned  the  proteins. 


350  PHYSICAL  PROPERTIES 

This  phenomenon  was  investigated  more  fully  by  Schulz  and 
Zsigmondy  (10),  who  define  the  number  of  milligrams  of  a  colloid 
which  is  just  insufficient  to  prevent  10  cc.  of  a  colloidal  gold  solu- 
tion from  showing  a  change  in  color  after  the  addition  of  1  cc. 
of  10  per  cent  NaCl  solutions  as  the  "  gold-number  "  of  the  colloid. 
It  appears  to  be  highly  characteristic  for  certain  proteins.  The 
following  are  the  values  determined  by  Schulz  and  Zsigmondy  for 
the  various  proteins  contained  in  or  derived  from  egg-white: 

Globulin 0.02-0.05 

Ovomucoid 0 . 04-0 . 08 

Crystallized  egg-albumin 2 . 00-8 . 00 

Other  (con-)  albumin 0 . 03-0 . 05 

Alkali-albuminate 0.006-0.04 

The  great  difference  between  the  crystalline  albumin  "  gold- 
number"  and  that  of  the  other  proteins  in  egg-white  made  it 
possible  to  recognize  the  presence  of  very  small  quantities  of 
colloidal  contamination  in  the  egg-albumin.  Schulz  and  Zsig- 
mondy were  able  to  show  by  this  method  that  egg-albumin  must 
be  recrystallized  from  three  to  six  times  to  remove  appreciable 
quantities  of  such  contaminations. 

The  determination  of  the  "  gold-number "  of  cerebro-spinal 
fluid  has  been  found  to  be  of  very  great  value  in  the  differential 
diagnosis  of  the  various  forms  of  syphilitic  sequelae,  the  modifi- 
cations observed  being  attributable  to  the  marked  increase  in 
globulin-content  which  accompanies  and  characterizes  certain  of 
these  conditions. 

LITERATURE   CITED 

(1)  Alsberg,  C.  L.,  and  Hedblom,  C.  A.,  Journ.  Biol.  Chem.,  6  (1909),  p. 

483. 

(2)  Arrhenius,  S.,  Zeit.  f.  physik.  Chem.,  1  (1887),  p.  285. 

(3)  Arrhenius,  S.,  "Immunochemistry,"  New  York  (1907). 

(4)  Barcroft,  J.,  and  Hill,  A.  V.,  Journ.  of  Physiol.,  39  (1910),  p.  411. 

(5)  Bayliss,  W.  M.,  Proc.  Roy.  Soc.,  London,  81  (1901),  p.  269. 

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CHAPTER  XIV 

OPTICAL  PROPERTIES   OF   PROTEIN   SOLUTIONS* 

1.   The  Specific  Rotatory  Power  of  the  Proteins.  —  It  was 

observed  by  Hoppe-Seyler  (16)  (18)  that  the  majority  of  animal 
proteins,  in  aqueous  solutions,  rotate  the  plane  of  polarized  light 
to  the  left,  and  he  measured  the  specific  rotatory  powers  of  several 
of  the  animal  proteins.  These  investigations  were  continued  by 
Fredericque  (7)  and  Ktihne  (26)  who  proposed  employing  the 
specific  rotatory  power  of  a  protein  as  a  means  of  characterizing 
it  and  establishing  its  identity.  The  results  which  were  obtained 
by  these  observers  have  been  greatly  extended  and  amplified  by 
a  large  number  of  observers  who  have  investigated  this  physical 
property  of  a  variety  of  proteins.  It  appears  that  all  of  the 
naturally  occurring  proteins,  excepting  the  nucleo-proteins  and 
haemoglobin,  rotate  the  plane  of  polarized  light  to  the  left.  The 
following  are  among  the  results  which  have  been  obtained  by 
observers  who  have  worked  with  proteins  of  animal  origin: 

TABLE  I 


Protein 

Observer 

Nature  of  solution 

Degrees 

Serum  albumin  
Serum  albumin                  

Hoppe-Seyler  (20) 
Fredericque  (7) 

in  water 

-56.00 
—57  30 

Serum  albumin  
Serum  albumin  

Serum  albumin  

Starke  (62) 
Sebelien  (59) 

Maximovitsch  (31) 

dialyzed     solution 
in  distilled  water 

-60.05 
-60.  10  to  -62.  60 

-47.47 

Egg  albumin  

F.  G.  Hopkins  (15) 

dilute   ammonium 

-30.70 

Egg  albumin 

Osborne  and  Camp- 

sulphate 
water 

—28  60  to  —30  80 

Lact-albumin  .'  

bell  (38) 
Sebelein  (59) 

dialyzed  solution  in 

-36.  40  to  36.  98 

a-crystallin  from  the  crystalline 
lens     .                        

Morner  (33) 

distilled  water 
neutral  solution  in 

-46  90 

dilute  ammonia 

*  The  opalescence  of  protein  solutions  has  already  been  discussed  (Chap 
XIII,  8). 

355 


356 


PHYSICAL  PROPERTIES 


TABLE  I.  —  (Continued) 


Protein 

Observer 

Nature  of  solution 

Degrees 

/3-crystallin  from  the  crystalline 
lens                                   .   ..   . 

Morner  (33) 

dialyzed     solution 

—43  30 

Fibrinogen  
Casein  
Casein  

Mittelbach  (32) 
Hoppe-Seyler  (20) 
Hoppe-Seyler  (20) 

in   dilute   acetic 
acid 
dilute  salt  solution 
neutral  solution 
faintly  alkaline 

-52.50 
-80.00 
—76  00 

Casein 

Hoppe-Seyler  (20) 

strongly  alkaline 

—91  00 

Serum  globulin 

Fredericque  (7) 

dilute  saline  solu- 

—47 80 

Ovomucoid                    

Osborne  and  Camp- 

tion     of     mixed 
globulins 

—61  10  to  —61  38 

Alkali-albumose  from  egg-albu- 
min   
Pepsin-peptone  from  fibrin  
Pepsin-peptone  from  fibrin  

Pepsin-glutin  peptone  

bell  (38) 

Maas  (30) 
Borkel  (3) 
Borkel  (3) 

Scheermesser  (55) 

water 
12.5  per  cent  am- 
monia 

-49.40 
-36.36 
-39.42 

-77.  08  to  -77.  81 

a-antipeptone,  by  tryptic  diges- 
tion from  fibrin 

Muller  (34) 

water 

—24  50 

0-antip«ptone  by  tryptic  diges- 
tion from  fibrin  
a-antipeptone  out  of  Witte's  pep- 
tone   
Antipeptone  from  gelatin  
Globin  from  haemoglobin  

Muller  (34) 

Siegfried  (60) 
Kruger  (25) 
Gamgee  and  Hill  (9) 

water 

water 
water 
weakly  acid  watery 

-32.40 

-18.  45  to  -19.69 
-100.80 
-65.50 

Globin  from  hsemoglobin  
Haemoglobin 

GamgeeandHill(9) 
Gamgee  and  Hill  (9) 

alcoholic  solution 
solution   in   dilute 
acetic  acid 
water 

-54.20  (red  light) 
+10  40  (red  light) 

Nucleo-histone  from  thymus  — 
ct-nucleo-protein  from  pancreas  .  . 
Nuclein  from  pancreas  

Nucleo-protein     of     suprarenal 
glands  

Gamgee    and    Jones 
(10) 
Gamgee    and    Jones 
(10) 
Gamgee    and    Jones 
(10) 
Gamgee    and    Jones 
(10) 

dilute  ammonia 
dilute  ammonia 
water 
water 

+37.50  (red  light) 

+37.  50  to  +38.  10 
(red  light) 
+64.40  (red  light) 

+48.  10  (red  light) 

ROTATORY  POWER 


357 


The  following  results  *  are  those  which  have  been  obtained  by 
observers  who  have  worked  with  vegetable  proteins. 


TABLE  II 


Protein 

Observer 

Nature  of  solution 

Degrees 

Edestin,  hemp-seed  
Edestin,  hemp-seed  
Edestin,  hemp-seed  
Excelsin,  Brazil-nut  

Chittenden  and  Mendel 
Alexander 
Osborne  and  Harris 
Alexander 

10%  NaCl 
10%  NaCl 
10%  NaCl 
10%  NaCl 

-  43.48 
-  41.5 
-  41.7 
-  40  5 

Excelsin,  Brazil-nut  
Globulin  flax-seed 

Osborne  and  Harris 
Alexander 

10%  NaCl 
10%  NaCl 

-  42.94 
—  38  5 

Globulin,  flax-seed 

Osborne  and  Harris 

10%  NaCl 

—  43  53 

Globulin,  squash-seed 

Osborne  and  Harris 

10%  NaCl 

—  38  32 

Amandin,  almond 

Osborne  and  Harris 

10%  NaCl 

—  56  44 

Corylin,  hazel-nut  .  .           .... 

Osborne  and  Harris 

10%  NaCl 

—  43  09 

Juglansin,  English  walnut  
Juglansin,      American      black 
walnut  

Osborne  and  Harris 
Osborne  and  Harris 

10%  NaCl 
10%  NaOl 

-  45.21 
—  44  43 

Juglansin,  butternut  
Phaseolin,  kidney-bean  

Osborne  and  Harris 
Osborne  and  Harris 

10%  NaCl 
10%  NaCl 

-  45.40 
—  41.46 

Legumin,  horse-bean  

Osborne  and  Harris 

10%  NaCl 

—  44.09 

Ricin  castor-bean 

Osborne,  Mendel  and  Harris 

water 

—  28  85 

Gliadin  wheat 

Osborne  and  Harris 

80%  alcohol 

—  92  28 

dliadin,  wheat 

Kjeldahl 

55%  alcohol 

—  92  0 

Gliadin,  wheat.  ..                .    . 

Kjeldahl 

glacial  acetic  acid 

—  81  f 

Gliadin,  wheat  

Kjeldahl 

5.1%  acetic  acid 

—111  0 

Gliadin,  wheat  
<iliadin,  wheat  
Gliadin,  wheat  
Gliadin,  wheat  

Kjeldahl 
Mathewson 
Mathewson 
Mathewson 

phenol 
70%  methyl  alcohol 
70%  ethyl  alcohol 
60%  ethyl  alcohol 

-130.0 
-  95.65 
-  91.95 
—  96  66 

Gliadin  wheat 

Mathewson 

50%  ethyl  alcohol 

—  98  45 

Gliadin,  wheat 

Mathewson 

60%  propyl  alcohol 

—  101  10 

Gliadin,  wheat 

Mathewson 

70%  phenol 

—123  15 

Gliadin,  wheat 

Mathewson 

phenol,  anhydrous 

—131  77 

Gliadin,  wheat                 ...   . 

Mathewson 

paracresol 

—  121  00 

Gliadin,  wheat  
Gliadin,  wheat  

Mathewson 
Mathewson 

benzyl  alcohol 
glacial  acetic  acid 

-  53.10 
—  78  60 

Gliadin  a,  wheat  

Lindet  and  Ammann 

70%  alcohol 

—  81  6 

Gliadin  ft,  wheat  

Lindet  and  Ammann 

70%  alcohol 

—  95  0 

Gliadin,  rye  

Kjeldahl 

55%  alcohol 

—  121  0 

Gliadin,  rye  

Kjeldahl 

"dilute  acid" 

—  144  0 

Gliadin  rye 

Kjeldahl 

glacial  acetic  acid 

—  105  0 

Gliadin,  rye 

Kjeldahl 

phenol 

—  157  0 

Gliadin,  rye 

Lindet  and  Ammann 

70%  alcohol 

—  87  8 

Gliadin,  barley  

Lindet  and  Ammann 

70%  alcohol 

—  87  8 

Hordein,  barley  
Hordein,  rye  
Zein  a,  maize  
Zein  /3  maize 

Lindet  and  Ammann 
Lindet  and  Ammann 
Lindet  and  Ammann 
Lindet  and  Ammann 

70%  alcohol 
70%  alcohol 
70%  alcohol 
70%  alcohol 

-137.5 
-137.5 
-  29.6 
—  40  0 

Zein,  maize 

Osborne  and  Harris 

90%  alcohol 

—  28  0 

Zein,  maize 

Kjeldahl 

75%  alcohol 

—  35  0 

Zein,  maize 

Kjeldahl 

glacial  acetic  acid 

—  28  0 

*  Cited  after  T.  B.  Osborne  (37),  which  consult  for  references  to  the 
literature. 


358  PHYSICAL  PROPERTIES 

From  these  results  it  is  clear  that  the  nature  of  the  solvent  plays 
a  very  considerable  part  in  determining  the  rotatory  power  of  pro- 
teins. Furthermore,  it  would  appear  that  as  a  rule  the  salts  which 
proteins  form  with  acids  and  bases  differ  very  markedly  in  rotatory 
power  from  the  free  protein  (3)  (20)  (60)  (1)  (4)  (6)  (39).  In  an 
analogous  manner  the  rotatory  powers  of  the  salts  which  the  simple 
amino-acids  form  with  acids  and  bases  differ  very  markedly  from 
those  of  the  free  acids  (40).  As  a  rule  too  little  attention  has 
been  paid  to  this  fact  by  investigators  who  have  sought  to  char- 
acterize the  proteins  by  their  rotatory  powers.  It  is  evident  that 
the  specific  rotatory  power  of  a  protein  cannot  be  regarded  as 
characteristic  of  it  until  the  nature  of  the  protein  salt  and  of  the 
solvent  employed  are  rigidly  defined. 

According  to  Alexander  (1)  the  rotatory  power  of  a  dissolved 
protein  is  considerably  affected  by  exposure  of  the  solution  to  a 
high  temperature  for  a  period  preceding  the  measurement.  It 
must  not  be  forgotten,  however,  that  partial  hydrolysis  of  the 
protein  may  play  a  very  considerable  part  in  bringing  about  this 
change. 

2.  The  Absorption  of  Light  by  Protein  Solutions.  —  The 
peculiar  absorption-spectra  of  solutions  of  haemoglobin  and  its 
colored  derivatives  have  been  very  extensively  studied  (17)  (19) 
(63)  (21)  (22)  (36)  (5)  (58)  (14).  From  the  results  of  these  in- 
vestigations it  is  evident  that  the  absorption-bands  (a  and  0)  in 
the  visible  spectrum  which  are  caused  by  these  solutions  are 
primarily  attributable  to  the  haematin-  and  not  to  the  distinctively 
protein  moiety  of  the  haemoglobin  molecule.  In  addition  to 
these  bands,  however,  the  absorption-spectrum  of  haemoglobin 
and  its  derivatives  reveals  another  absorption-band  on  the  extreme 
edge  of  the  violet  end  of  the  visible  spectrum  (61)  (2)  (8).  Ac- 
cording to  Gamgee  (8)  this  absorption-band  is  given  not  only  by 
solutions  of  haemoglobin  but  also  by  solutions  of  haematin;  he 
therefore  attributes  it  to  the  haematin,  and  not  to  the  protein 
moiety  of  the  haemoglobin  molecule.  According  to  Soret,  how- 
ever, an  absorption-band  on  the  extreme  edge  of  the  ultra-violet 
can  be  demonstrated,  by  employing  a  fluorescent  screen,  in  the 
absorption-spectra  of  solutions  of  a  large  variety  of  proteins, 
among  which  may  be  mentioned  egg-albumin,  mucin  (from 
snails),  casein,  globulin  and  syntonin.  This  band  is  followed,  on 
the  ultra-violet  side,  by  a  region  of  especial  transparency.  Accord- 


REFRACTIVE  INDICES  359 

ing  to  Soret  it  is  unaffected  by  acids,  but  on  adding  NaOH  or 
ammonia  to  the  protein  solution  the  region  of  transparency  in 
the  ultraviolet  disappears  completely  and  the  band  of  absorption 
is  displaced  towards  the  less  refrangible  end  of  the  spectrum.  On 
neutralizing  the  solution  with  acid  the  original  absorption-spectrum 
is  restored.  A  similar  absorption-spectrum  is  yielded  by  solutions 
of  tyrosin  and  it  is  affected  in  a  similar  manner  by  alkalies.  Soret 
therefore  attributes  the  absorption-band  in  the  absorption  spectra 
of  protein  solutions  to  the  tyrosin  radical  which  the  proteins 
contain. 

Kober  (23)  has  recently  carried  out  a  spectrographic  examina- 
tion of  solutions  of  a  number  of  amino-acids  and  polyamino-acids. 
He  finds  that  the  aliphatic  amino-acids  display  only  general 
absorption  in  the  extreme  ultraviolet.  The  aromatic  acids, 
tyrosin  and  phenylalanin  do,  however,  show  definite  absorption- 
bands  in  the  ultra-violet  spectrum.  The  absorption-band  in  the 
ultraviolet  which  is  displayed  by  proteins  in  solution  is  therefore 
attributable  not  only  to  tyrosin  radicals,  as  Soret  supposed,  but 
also  in  some  measure  to  phenylalanin  radicals. 

Since  the  occurrence  of  any  photochemical  reaction  is  dependent 
upon  the  absorption  of  the  chemically  active  rays  by  some  con- 
stituent or  constituents  of  the  reacting  system  it  would  appear 
possible  that  the  markedly  toxic  action  of  ultraviolet  light  upon 
many  unicellular  organisms  may  be  dependent  upon  the  absorption 
of  the  ultraviolet  light-rays  by  the  proteins  of  the  organisms 
affected.  Strong  confirmation  of  this  view  is  afforded  by  the 
experiments  of  Harris  and  Hoyt  (12)  who  have  shown  that  if  ultra- 
violet light  be  passed  through  a  thin  layer  of  gelatin  or  peptone 
solution  its  toxicity  for  paramoeda  is  diminished  to  a  much 
greater  extent  than  by  passage  through  a  similar  layer  of  distilled 
water.  Solutions  of  urea,  sugar  or  alanin  do  not  confer  this  pro- 
tection, while  solutions  of  the  aromatic  amino-acids  protect  the 
organisms  very  markedly.  Leucin  likewise  confers  a  very  marked 
protection,  but  this  amino-acid  is  rapidly  decomposed  by  ultra- 
violet light,  yielding  colored  products,  and  the  decomposition  is  ac- 
companied by  an  increase  in  the  protective  power  of  the  solution. 

3.  The  Refractive  Indices  of  Protein  Solutions.  —  The  in- 
fluence of  dissolved  proteins  upon  the  refractive  indices  of  various 
solvents  has  been  especially  studied  by  Reiss  (41)  (43),  Herlitzka 
(13),  Schmidt  (56)  and  myself  (43-54). 


360  PHYSICAL  PROPERTIES 

Reiss  measured  the  change  in  the  refractive  indices  of  dilute 
salt  solutions  which  results  from  the  introduction  of  varying 
amounts  of  the  "  pseudoglobulins "  of  blood  serum.  His  pseudo- 
globulins  were  prepared  by  fractional  coagulation  with  ammonium 
sulphate  and  purified  by  prolonged  dialysis.  Fraction  I  (pseudo- 
globulin  I)  was  coagulated  at  32  to  36  per  cent  saturation  with 
ammonium  sulphate,  fraction  II  (pseudoglobulin  II)  was  coagu- 
lated at  36  to- 39  per  cent  saturation.  The  change  in  the  refractive 
index  of  the  solvent  due  to  the  introduction  of  these  proteins  was 
found  to  be  directly  proportional  to  the  quantity  of  protein  dis- 
solved in  it;  the  change  due  to  the  introduction  of  1  per  cent  of 
the  "pseudoglobulin  I "  being  0.00224  and  that  due  to  the  introduc- 
tion of  1  per  cent  of  the  " pseudoglobulin  II"  0.00230.  The 
difference  between  these  determinations  is  not  sufficient  to  con- 
stitute a  basis  for  distinction  between  the  two  globulin-fractions, 
since  it  is  not  greater  than  that  which  might  have  arisen  through 
experimental  error.  Reiss  also  measured  the  influence  of  other 
constituents  of  serum  (especially  crystallized  and  amorphous 
serum  albumin)  upon  the  refractive  indices  of  dilute  saline  solu- 
tions. 

I  have  amplified  and  confirmed  these  results  of  Reiss,  employing 
for  this  purpose  a  variety  of  proteins  (casein,  paranuclein,  ovo- 
mucoid,  ovovitellin,  serum  globulin,  gliadin,  etc.)  and  not  only 
aqueous  solvents  but  alcohol-water  mixtures,  acetone-water  mix- 
tures, etc.  A  large  number  of  determinations  upon  casein  in 
aqueous  solutions,  between  the  concentrations  which  it  is  feasible 
to  employ*  show  that  the  change  in  the  refractive  indices  of 
aqueous  solvents  due  to  the  introduction  of  this  protein  is  very 
accurately  proportional  to  its  concentration  (43)  (56).  Accord- 
ingly the  refractive  index  of  a  solution  of  this  protein  is  given  by: 

n  —  HI  =  a  X  c 

where  n  is  the  refractive  index  of  the  solution,  n\  that  of  the  solvent, 
c  the  percentage  of  casein  in  the  solution  and  a  a  constant  which 
is  characteristic  of  the  protein  (for  example,  casein)  which  is 

*  If  the  concentration  of  protein  be  too  small  (less  than  about  0.5  per  cent) 
the  experimental  error  of  the  determination  becomes  a  significant  proportion 
of  the  observed  difference  in  the  refractive  index  of  the  solvent,  due  to  the 
introduction  of  the  protein.  If  the  concentration  of  protein  be  too  large  then 
the  solutions  are  too  opaque  to  enable  an  exact  reading  to  be  obtained. 


REFRACTIVE  INDICES 


361 


employed  and  represents  the  change  in  the  refractive  index  of  the 
solvent  which  is  brought  about  by  dissolving  one  gram  of  the 
protein  in  100  cc. 
The  following  results  are  illustrative:* 

TABLE  III 
SolventO.lOATNaOH 


Concentration 
casein  per  cent 

Refractive  index 

a  for  nj  =  1.33444 

0.5 

1.3352 

0.00152 

1.0 

1.3360 

0.00156 

1.5 

1.3368 

0.00157 

2.0 

1.3375 

0.00153 

2.5 

1.3383 

0.00154 

3.0 

1.3390 

0.00152 

4.0 

1.3405 

0.00152 

5.0 

1.3420 

0.00151 

6.0 

1.3436 

0.00153 

In  the  first  column  of  the  table  is  given  the  amount  of  casein  in 
grams  which  was  dissolved  in  100  cc.  of  the  solution.  In  the 
second  column  is  given  the  refractive  index  of  the  solution  meas- 
ured at  20°  C.  In  the  third  is  given  the  value  of  the  constant  a 
calculated  from  the  above  formula  for  the  given  value  of  the 
constant  wi  (i.e.,  the  refractive  index  of  the  solvent,  previously 
determined  at  the  same  temperature). 

The  equation  n  —  HI  =  a  X  c  also  holds  good  for  solutions  of 
ovomucoid  in  water  (44),  of  paranuclein  in  JV/10  KOH  (45),  of 
potassium  serum  globulinate  (46),  of  potassium  casemate  in 
alcohol- water  mixtures  (47),  of  gliadin  in  various  solvents  (54)  and 
of  edestin  in  aqueous  solvents  (56). 

The  change  in  the  refractive  indices  of  aqueous  solvents  which 
is  brought  about  by  a  given  percentage  of  casein  is  independent  of 
the  nature  of  the  acid  or  base  with  which  the  casein  is  combined. 
This  is  shown  by  the  fact  that  this  change  is  the  same  whether  the 
casein  be  dissolved  in  dilute  KOH,  NaOH,  LiOH,  NH4OH, 
Sr(OH)2,  Ba(OH)2,  Ca(OH)2  or  HC1  solutions.  Also,  between 
0.01  N  and  0.1  N  it  is  independent  of  the  concentration  of  alkali 

*  In  all  of  these  determinations  a  Pulfrich  refractometer  was  employed, 
reading  the  angle  of  total  reflection  accurately  to  within  1',  and  a  sodium  flame 
was  employed  as  the  source  of  light. 


362  PHYSICAL  PROPERTIES 

which  is  employed  as  solvent.  Similarly,  the  value  of  a,  in  the 
above  equation,  is  the  same  for  " insoluble"  serum  globulin 
whether  this  protein  be  dissolved  in  TV/IO  KOH  or  in  N/40  HC1. 
The  influence  which  a  protein  exerts  upon  the  refractive  index  of 
its  solution  is  therefore  independent  of  the  nature  or  proportion  of 
acid  or  base  which  is  combined  with  it,  differing  in  this  respect  very 
strikingly  from  the  optical  rotatory  power  of  dissolved  protein, 
which,  as  we  have  seen,  is  very  intimately  dependent  upon  the 
nature  and  proportion  of  combined  inorganic  acid  or  base.  The 
reason  for  this  fact  is  readily  perceived  when  we  reflect  that  the' 
power  of  dissolved  protein  to  refract  light  is  a  function  of  the  space 
which  is  occupied  by  the  protein  molecules.  Now  the  molecular 
volume,  and,  indeed,  the  molecular  refractivity  is  an  additive 
function  of  the  atomic  volumes  (or  refractivities)  of  the  atoms 
which  together  make  up  the  molecule.  In  a  molecule  which 
contains  over  a  thousand  atoms,  as  a  protein  molecule  does,  the 
substitution  of  even  several  H  atoms  by  K  atoms  or  the  addition 
of  a  few  H  or  Cl  atoms  or  OH  groups  might  be  expected  not  to 
exert  an  appreciable  influence  upon  the  volume  or  refractive  power 
of  the  whole  molecule.  The  refractive  index  of  a  protein  solution 
also  remains  unaltered  by  hydrolysis,  upon  which  fact  I  have 
based  a  method  of  determining  the  comparative  activities  of 
trypsin  solutions  (50). 

From  the  investigations  of  Gladstone  and  Dale  (11)  and  of 

N  —  1 
Landolt  (27)  it  appears  that  the  expression  — -, — ,  where  N  is  the 

refractive  index  of  a  substance  and  d  its  density,  which  is  known 
as  the  specific  refractivity,  is  very  constant,  being  only  slightly 
dependent  upon  the  temperature.  Moreover  .each  particular 
substance  in  a  mixture  preserves  its  own  specific  refractivity 
nearly  unchanged;  hence  the  refractive  index  of  a  mixture  or  of 
a  solution  can  generally  be  readily  calculated  from  the  refractive 
indices  of  its  components.  Now  the  density  of  a  dilute  protein 
solution  is  always  very  nearly  that  of  water,  that  is  1,  so  that  the 
specific  refractivity  of  a  dilute  protein  solution  may,  with  a  toler- 
able approach  to  accuracy,  be  taken  as  n  —  1,  where  n  is  the  re- 
fractive index  of  the  solution.  Suppose  c  per  cent  of  protein  were 
to  be  dissolved  in  a  solvent  of  specific  refractivity  Ni  —  I  (for 
instance,  a  dilute  acid  or  alkali,  which  is  nearly  equal  in  density  to 
pure  water),  then,  if  Gladstone's  law  holds  good,  and  the  density 


REFRACTIVE  INDICES  363 

of  the  dissolved  protein  itself  be  d  (Cf.  Chap.  XIII,  section  11) 
the  refractive  index  of  the  mixture  should  be  given  by: 


whence 
and 


(ri      n_  W-l)(100-c)      (N-l)c   1 

100  100   "V 

100  (n  -1)  =  100  (Ni  -  1)  -  c  (l  -  i  -  Ni  +  %} 

\        a  a/ 


n  — 


100 


or  »T       /-       1       TIT    ,  -AT\    v 

n-A^fl  --_#!+_)_,  (j) 


which  is  identical  with  the  relation  n  —  HI  =  a  X  C  which,  as  we 
have  already  seen,  subsists  between  the  refractive  index  of  a  pro- 
tein solution  and  its  percentage  concentration.*  Hence  we  may 
conclude  that  for  solutions  of  proteins  in  aqueous  solvents  Gladstone's 
law  of  mixtures  holds  good.  The  constant  a  is  obviously  given  by 

y^.  (1  —  -3  —  Ni  +  -T  j  .  For  solutions  of  casein  containing  be- 
tween 0.25  and  6.00  per  cent  the  average  value  of  a  is  0.00125. 
Hence,  by  exterpolation,  the  refractive  index  of  the  pure  protein 
should  be  1.675,  taking  the  refractive  index  of  water  as  1.333  and 
the  density  of  dissolved  casein  as  1.39  (Cf.  Chap.  XIII,  section  11). 
For  serum  globulin  this  constant  is  1.774.  The  refractive  index 
of  the  pure  protein  does  not  readily  admit  of  direct  determination 
in  these  cases.  The  protamin  salts,  clupein  sulphate  and  salmin 
sulphate  can,  however,  readily  be  obtained  in  a  fluid  (oily)  con- 
dition. Kossel  (24)  has  determined  the  refractive  indices  of  these 
fluids  and  finds  them  to  be,  respectively,  1.443  and  1.442,  figures 
which  are  of  the  same  order  of  magnitude  as  the  above  estimates. 
If  the  density  of  the  solvent  be  d'  instead  of  1  and  c  be  small,  so 
that  the  density  of  the  solution  is  very  nearly  that  of  the  solvent, 
then  equation  (i)  becomes: 

n~Ni  =  (1-j-Ni+jN)m          <H> 

*  From  this  it  would  appear  that  the  value  of  a  should  vary  with  the  re- 
fractive index  even  of  a  dilute  solution  of  a  base  or  acid  which  is  not  exactly 
the  same  as  that  of  water,  that  is,  with  the  nature  and  concentration  of  the 
(dilute)  acid  or  base  employed  as  a  solvent.  It  will  be  observed,  however, 
that  in  the  experimental  values  of  a  only  a  one-hundredth  part  of  the  difference 
between  the  refractive  index  of  the  acid  or  alkali  solution  and  that  of  water 
appears.  This  quantity,  for  the  solutions  employed,  is  far  too  small  to  be 
detectable. 


364  PHYSICAL  PROPERTIES 

which  is  the  same  form  as  (i) .  Obviously,  however,  in  a  solution  of 
a  protein  in  a  solvent  the  density  or  refractivity  of  which  departs 
at  all  widely  from  those  of  water,  although  the  law  n  —  n\  =  a  X  c 
might  be  expected  to  hold  good,  the  constant  a  would  not  have 
the  same  value  as  in  aqueous  solvents.  In  other  words,  in  non- 
aqueous  solvents  the  law  n  —  n\  =  a  X  c  should  hold  good,  but, 
for  a  given  protein  the  value  of  a  should  vary  with  the  nature  of 
the  solvent  depending  upon  its  refractivity  and  its  density.  In 
satisfactory  correspondence  with  this  deduction  we  find  that  the 
law  n  —  HI  =  a  X  c  holds  good  for  solutions  of  potassium  casein- 
ate  in  alcohol-water  mixtures  containing  from  0  to  75  per  cent  of 
alcohol,  for  solutions  of  serum-globulin  in  alcohol-water  mixtures, 
and  for  solutions  of  gliadin  in  a  variety  of  solvents.  As  we  shall 
see,  however,  although  the  value  of  a  for  a  given  protein  varies 
with  the  nature  of  the  solvent  in  the  sense  demanded  by  the  above 
theory,  this  variation  cannot  be  quantitatively  expressed  by  the 

formula  100  a  =  N  ^  -Ni  +  1  -  ~ 
a  a 

In  estimating  the  accuracy  with  which  a  can  be  determined  in 
the  equation  n  —  n\  =  a  X  c,  it  is  essential  to  recollect  that  the 
experimental  error  in  determining  the  angle  of  total  reflection  with 
the  Pulfrich  Refractometer  is  ±1'.  This,  for  the  instruments 
which  I  have  employed,  corresponds  to  an  error  of  ±0.00009  in 
the  determination  of  the  refractive  index  of  a  given  solution.  Now 
it  is  obvious  that  since  the  absolute  error  in  the  determination  of 
n  —  H!  is  the  same,  the  error  in  the  determination  of  a  must  be 
less  in  proportion  to  the  magnitude  of  c. 

In  order  to  assign  to  each  determination  its  due  weight  in  the 
estimation  of  the  mean  value  of  a  for  any  solvent  we  must  therefore 
add  together  all  of  the  observed  values  of  n  —  n\  and  divide  this 
sum  by  the  sum  of  the  concentrations  employed.  This  procedure 
has  been  adopted  in  my  own  estimates,  cited  below.  The  follow- 
ing are  the  values  of  a  for  various  proteins  and  solvents  which 
have  so  far  been  ascertained.  The  influence  of  temperature  upon 
the  magnitude  of  a  appears  to  be  very  slight.  In  fact  for  casein  I 
have  found  the  value  of  a  to  be  the  same,  within  the  experimental 
error,  at  40  degrees  as  it  is  at  20  degrees.  Hence  the  temperatures 
at  which  the  observations  were  made  are  not  specified  in  the 
accompanying  table;  they  lay,  in  every  case,  however,  between 
ordinary  room-temperatures  and  24  degrees. 


REFRACTIVE  INDICES 


365 


iO"OiO»OiOiOiOtO 


OOOOOOOO 


.  fl  d  d  S3  d 
i-HOOOOO 
o>  02  IB  02  °2 

Hill 


OOOOOOO 


OO 


8  1  » 


ooooo 

-H-H-H-H-H  -H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H 

•  05  C3  05  t-  CO  TJH 


oooooooooooooooooooooooooooo 


>  V 

00   _|| 


_   03 

o  fl 

O 


nr 

o  o 


|lllHll|l|o|l 


d  a  a  a 


s  a  a 


llllll=JJJfl- 


<1 

•  is  ;  ;  ;  ;.s.a 

" 

:  :  S  i  i  ;  '"ii'i. 

S'd'S^??^????  ~ 

*rr   MGc3c3c3c30g«TOC3 


366  PHYSICAL  PROPERTIES 

Calculating  the  theoretical  values  of  a,  on  the  assumption  that 
Gladstone's  law  of  mixtures  holds  good  for  solutions  of  protein  in 
any  solvent  we  at  once  find  that  although  a  varies  with  the  nature 
of  the  solvent  in  the  sense  demanded  by  Gladstone's  law,  yet  the 
law  does  not  hold  good,  for  different  solvents,  with  any  approach 
to  quantitative  exactitude.  Thus  the  calculated  value  of  a  for 
solutions  of  casein  in  50  per  cent  alcohol  is  0.00095,  assuming  the 
density  of  casein  dissolved  in  50  per  cent  alcohol  to  be  the  same 
as  that  of  casein  dissolved  in  water,  while  if  we  assume  the 
density  of  casein  dissolved  in  50  per  cent  alcohol  to  be  that  of  un- 
dissolved  casein  (Cf.  Chap.  XIII,  section  11)  the  calculated  value 
of  a  becomes  0.00121;  the  experimental  value,  however,  is  0.00149; 
the  calculated  value  of  a  for  insoluble  serum-globulin  in  50  per  cent 
alcohol  is  0.00169,  the  experimental  value  is  0.00119.  Hence  Glad- 
stone's law  of  mixtures  holds  good  for  solutions  of  protein  in  a 
specified  solvent,  but  no  longer  holds  good  if  we  vary  the  nature 

N  —  1 
of  the  solvent.     Now  it  is  a  characteristic  of  the  quantity  — -= — 

a 

that  although  it  is  independent  of  temperature  and  of  concentra- 
tion, it  varies  when  the  state  of  aggregation  varies  (35).  We  may 
conclude,  therefore,  as  we  have  had  occasion  to  indicate  before, 
that  the  physical  condition  (number  of  associated  water  molecules, 
degree  of  dispersion,  etc.)  of  proteins  in  alcohol-water  mixtures  and 
other  non-aqueous  solvents  is  not  the  same  as  it  is  in  water.* 

Comparing  the  above  cited  determinations  by  Robertson  and 
Greaves,  of  the  refractive  indices  of  gliadin  in  various  solvents, 
with  Mathewson's  determination  of  the  rotatory  power  of  gliadin 
in  various  solvents,  it  is  evident  that,  as  might  have  been  expected, 
there  is  no  correspondence  between  the  effects  of  different  solvents 
upon  these  two  physical  properties  of  dissolved  protein. 

It  will  be  observed  that  the  value  of  a  for  solutions  of  gliadin  in 
75  per  cent  phenol  is  negative.  This  is  due  to  the  fact  that  the 
refractive  index  of  75  per  cent  phenol  is  nearly  equal  to  that  of 
gliadin  itself,  and  its  density  is  greater  so  that  a  mixture  of  the  two 
substances  has  a  refractive  index  which  is  less  than  that  of  the  75 
per  cent  phenol  employed  as  solvent. 

The  influence  of  temperature  upon  the  refractive  indices  of 
protein  solutions  has  been  determined  by  myself  and  by  Herlitzka 
(13).  As  stated  above,  I  find  the  effect  of  temperature  upon  the 

*  Cf .  Chaps.  I  and  XIII. 


REFRACTIVE  INDICES  367 

value  of  a  for  solutions  of  caseinates  in  water  to  be  very  small.  In 
fact,  if  allowance  be  made  for  the  alteration  in  the  refractive  index 
of  the  water  with  varying  temperature,  I  have  been  unable  to 
observe  any  alteration  in  a,  distinctly  greater  than  the  possible 
experimental  error,  between  20  and  40  degrees.  According  to 
Herlitzka,  however,  if  egg-albumin  be  employed,  a  distinct  in- 
fluence of  temperature  upon  the  refractivity  of  the  protein  itself 
can  be  observed.  He  interprets  his  results  in  the  light  of  the 
Lorentz-Lorentz  modification  of  Gladstone's  law  of  mixtures  (28) 
(29). 

The  utilization  of  the  refractometric  method  for  determining 
the  concentrations  of  various  proteins  in  solutions  and  in  body- 
fluids  has  been  discussed  in  Chap.  III. 

LITERATURE   CITED 

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340. 

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p.  10. 

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(1858),  p.  8;  153  (1863),  p.  316. 

(12)  Harris,  F.  I.,  and  Hoyt,  H.  S.,  "Science,"  N.  S.,  46  (1917),  p.  318. 

(13)  Herlitzka,  A.,  Zeit.  f.  chem.  und  der  Kolloide,  7  (1910),  p.  251. 

(14)  Heubner,  W.,  and  Rosenberg,  H.,  Biochem.  Zeit.,  38  (1912),  p.  345. 

(15)  Hopkins,  F.  Gowland,  Journ.  of  Physiol.,  25  (1900),  p.  306. 

(16)  Hoppe-Seyler,  F.,  Virchow's  Arch.,  11  (1857),  p.  552. 

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(18)  Hoppe-Seyler,  F.,  Zeit.  f.  chem.  und  Pharm.,  7  (1864),  p.  737. 

(19)  Hoppe-Seyler,  F.,  Med.-Chem.  Untersuch.  (1867),  p.  169. 

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(21)  Hufner,  G.,  Journ.  f.  prakt.  Chem.  (2),  22  (1880),  p.  362. 

(22)  Hufner,  G.,  Arch.  f.  (Anat.  und)  Physiol.  (1890),  p.  1;  (1894),  pp.  130 

and  209;    (1895),  p.  213;   (1901),  Suppl.,  p.  187. 

(23)  Kober,  P.  A.,  Journ.  Biol.  Chem.,  22  (1915),  p.  433. 


368  PHYSICAL  PROPERTIES 

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(25)  Kruger,  T.  H.,  Zeit.  f.  physiol.  Chem.,  38  (1903),  p.  320. 

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(27)  Landolt,  H.,  Pogg.  Ann.,  123  (1864),  p.  595. 

(28)  Lorentz,  H.  A.,  Weid.  Ann.,  9  (1880),  p.  641. 

(29)  Lorentz,  L.,  Weid.  Ann.,  11  (1880),  p.  70. 

(30)  Maas,  O.,  Zeit.  f.  physiol.  Chem.,  30  (1900),  p.  61. 

(31)  Maximovitsch,  S.,  Journ.  Russ.  phys.  Chem.  Soc.,  6  (1901),  p.  460; 

cited  after  Maly's  Jahresbr.  f.  Tierchem.,  31,  p.  35. 

(32)  Mittelbach,  F.,  Zeit.  f.  physiol.  Chem.,  19  (1894),  p.  289. 

(33)  Morner,  C.  T.,  Zeit.  f.  physiol.  Chem.,  18  (1893),  p.  61. 

(34)  Miiller,  F.,  Zeit.  f.  physiol.  Chem.,  38  (1903),  p.  265. 

(35)  Nernst,  W.,  "Theoretical  Chemistry,"  Transl.  4th  German  Edn., 

New  York  (1904),  p.  307. 

(36)  von  Noorden,  C.,  Zeit.  f.  physiol.  Chem.,  4  (1879),  p.  9. 

(37)  Osborne,  T.  B.,  "The  Vegetable  Proteins,"  London,  1909. 

(38)  Osborne,  T.  B.,  and  Campbell,  G.  F.,  Journ.  Amer.  Chem.  Soc.,  22 

(1900),  p.  422. 

(39)  Pauli,  W.,  Samec,  M.,  and  Strauss,  E.,  Biochem.  Zeit.,  59  (1914),  p. 

470. 

(40)  Plimmer,  R.  H.  A.,  "The  Chemical  Constitution  of  the  Proteins," 

London  (1908),  Part  I,  p.  79. 

(41)  Reiss,  E.,  Arch.  f.  exper.  Path,  und  Pharm.,  51  (1903),  p.  18. 

(42)  Reiss,  E.,  Beitr.  z.  chem.  Physiol.  und  Path.,  4  (1904),  p.  150. 

(43)  Robertson,  T.  Brailsford,  Journ.  Physical  Chem.,  13  (1909),  p.  469. 

(44)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  7  (1910),  p.  359. 

(45)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  8  (1910),  p.  287. 

(46)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  8  (1910),  p.  441. 

(47)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  8  (1910),  p.  507. 

(48)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  11  (1912),  p.  179. 

(49)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  11  (1912),  p.  307. 

(50)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  12  (1912),  p.  23. 

(51)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  13  (1912),  p.  325. 

(52)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  13  (1913),  p.  455. 

(53)  Robertson,  T.  Brailsford,  Journ.  Biol.  Chem.,  13  (1913),  p.  499. 

(54)  Robertson,  T.  Brailsford,  and  Greaves,  J.  E.,  Journ.  Biol.  Chem.,  9 

(1911),  p.  181. 

(55)  Scheermesser,  W.,  Zeit.  f.  physiol.  Chem.,  37  (1903),  p.  363. 

(56)  Schmidt,  C.  L.  A.,  Journ.  Biol.  Chem.,  23  (1915),  p.  487. 

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(62)  Starke,  K.  V.,  Upsala  lakareforening  forhandlinger,  16,  cited  after 

Maly's  Jahresber.  f.  Tierchem.,  11  (1881),  p.  17. 

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PART  IV 
CHEMICAL  DYNAMICS  IN  PROTEIN  SYSTEMS* 

*  Any  survey  of  this  subject  must,  of  course,  involve  an  incidental  exposi- 
tion of  the  properties  and  behavior  of  the  enzymes,  to  which  reference  must 
necessarily  be  made.  Nevertheless  any  attempt  to  enter  fully  into  a  discussion 
of  the  properties  of  enzymes  would,  of  course,  lead  us  into  regions  quite  for- 
eign to  the  scope  of  this  work.  For  exhaustive  treatments  of  the  general 
subject  of  enzyme-action  the  reader  is  referred  to  the  works  of  Oppenheimer 
(15),  Bredig  (8),  Taylor  (19),  Vernon  (20),  Euler  (9)  and  Bayliss  (7). 


CHAPTER  XV 
THE  HYDROLYSIS  OF  THE  POLYPEPTIDS 

1.  The  Hydrolysis  of  Polypeptids  by  Proteolytic  Enzymes.  — 

By  the  aid  of  the  various  hydrolysing  agencies  the  synthetic  poly- 
peptids  are  capable  of  being  split,  with  successive  additions  of 
the  elements  of  water,  into  their  constituent  amino-acids.  Among 
these  hydrolysing  agencies  must  be  reckoned  a  number  of  the 
protein-splitting  enzymes  which  are  found  in  a  variety  of  tissues, 
tissue-extracts  and  secretions. 

The  action  of  the  enzymes  of  the  pancreas  (trypsin  and,  pre- 
sumably, others)  upon  the  dipeptids  was  first  investigated  by 
Fischer  and  Bergell  (12)  who  acted  upon  solutions  of  various 
dipeptids  with  pancreas-extract.  They  found  that: 


glycyl-glycin 

/3-naphthalenesnlphoglycyl-d-alanin 
/3-naphthalenesulpho-d-alanyl-glycin 
Di-/3-naphthalenesulphotyrosyl-dl-leucin 
while 

j8-naphthalenesulphoglycyl-l-tyrosin 

/3-naphthaleneglycyl-dl-leucin 

carboxethyl-glycyl-dl-leucin 

glycyl-1-tyrosin 

leucyl-alanin 

alanyl-leucin 

leucyl-leucin 


were  not  hydrolysed. 


were  hydrolysed. 


In  the  course  of  these  investigations  the  exceedingly  interest- 
ing fact  was  encountered  that  the  racemic  compounds  are  hydro- 
lysed asymmetrically  by  the  pancreas  extract;  thus  only  1-leucin 
was  obtained  by  the  action  of  the  extract  upon  carboxethyl- 
glycyl-d-1-leucin,  the  remainder  (containing  d-leucin)  not  being 
acted  upon.  Now  l-leucin  is  the  naturally  occurring  variety,  the 
form,  that  is,  which  is  found  in  the  proteins. 

These  investigations  were  greatly  extended  by  Fischer  and 
Abderhalden  (11)  who  employed  a  pancreatic  juice  prepared 
by  Pavlov  from  a  pancreatic  fistula  and  activated  by  entero- 

371 


372 


CHEMICAL  DYNAMICS 


kinase  obtained  from  the  succus  entericus  of  the  duodenum. 
The  result  of  these  investigations  was  to  show  that  the  synthetic 
polypeptids  can  be  divided  into  two  distinct  classes,  members 
of  the  one  class  being  hydrolysed  or  (if  racemic)  partially  hydro- 
lysed  by  the  trypsin;  members  of  the  other  class  not  being 
hydrolysed  by  trypsin.  The  various  polypeptids  employed  by 
Fischer  and  Abderhalden  were  distributed  between  these  two 
classes  as  follows: 


Hydrolysed 

*  Alanyl-glycin 

*  Alanyl-alanin 

*  Alanyl-leucin  A 

*  Leucyl-isoserin  A 
Glycyl-1-tyrosin 
Leucyl-1-tyrosin 

*  Alanyl-glycyl-glycin 

*  Leucyl-glycyl-glycin 

*  Glycyl-leucyl-alanin 

Not  Hydrolysed 
Glycyl-alanin 
Glycyl-glycin 
Alanyl-leucin  B 
Leucyl-alariin 
Leucyl-glycin 
Leucyl-leucin 
Aminobutyryl-glycin 
Aminobutyryl-aminobutyric  acid  A 
Aminobutyryl-aminobutyric  acid  B 
Valyl-glycin 


Alanyl-leucyl-glycin 

Dialanyl-cystin 

Dileucyl-cystin 

Tetraglycyl-glycin 

Triglycyl-glycin  ester 

d-alanyl-d-alanin 

d-alanyl-1-leucin 

1-leucyl-l-leucin 

1-leucyl-d-glutamic  acid 


Glycyl-phenylalanin 

Leucyl-prolin 

Diglycyl-glycin 

Triglycyl-glycin 

Dileucyl-glycyl-glycin 

d-alanyl-1-alanin 

1-alanyl-d-alanin 

1-leucyl-d-leucin 

d-leucyl-1-leucin 


The  compounds  which  are  marked  by  asterisks  were  racemic. 
Of  all  these  the  hydrolysis  proved  to  be  asymmetric  and  only  the 
naturally-occurring  stereo-isomer  was  attacked. 

There  is  thus  a  remarkable  correlation  between  the  ability  of 
these  enzymes  to  attack  certain  amino-acid  unions  and  the  occur- 
rence of  these  unions  among  the  natural  proteins  which  these 
enzymes  attack  in  the  ordinary  course  of  events.  As  we  shall 
see  in  the  following  chapter  there  are  many  excellent  reasons 
for  believing  that  the  action  of  the  proteolytic  enzymes,  like 
that  of  many  of  the  inorganic  catalysors,  is  dependent  upon  the 
formation  of  intermediate  compounds  of  the  enzyme  with  the 
substrate.  It  appears  highly  probable,  therefore,  that  the  enzyme 


HYDROLYSIS  OF  POLYPEPTIDS  373 

is  itself  possessed  of  an  optical  structure  which  fits  it  to  unite 
with  certain  stereo-isomers  and  only  with  them.  To  pursue 
Emil  Fischer's  classical  illustration,  the  enzyme  and  these  par- 
ticular stereo-isomeric  unions  fit  one  another  as  a  key  does  a  lock. 
The  position  is  therefore  this,  that  the  proteolytic  enzymes  are 
only  adapted  to  "fit"  certain  modes  of  union  between  amino- 
acids  and  that  these  are  precisely  the  modes  of  union  which  occur 
in  the  proteins  which  it  is  their  function  to  digest.  To  a  certain 
type  of  mind,  a  teleological  "explanation"  of  this  fact  would 
doubtless  prove  very  inviting.  But  the  biochemist  demands 
something  more  satisfactory  than  teleology,  or  even  than  the 
conveniently  comprehensive  nee-Darwinian  generalization  of 
"adaptation  through  survival  of  the  fittest."  Our  physico- 
chemical  view-point  inclines  us  to  suspect  the  existence  of  some 
physico-chemical  mechanism  which,  in  the  building  up  of  these 
bodies  has  necessarily  brought  about  their  mutual  adaptation  to 
and  dependence  upon  one  another.  The  nature  of  this  mechanism 
is,  I  believe,  not  far  to  seek.  In  Chap.  XVII  we  shall  see  that 
not  only  the  hydrolysis  but  also  the  synthesis  of  the  proteins  is 
brought  about  through  the  agency  of  enzymes,  and  that  the 
enzymes  which  bring  about  the  synthesis  of  the  proteins  are, 
in  all  probability,  but  slight  modifications  (probably  anhydrides) 
of  the  enzymes  which  bring  about  their  hydrolysis.  Synthesis, 
just  as  hydrolysis,  must  involve  the  formation  of  intermediate 
compounds  between  the  enzyme  and  the  substrate  (in  this  in- 
stance the  constituent  amino-acids  out  of  which  the  protein  is 
to  be  built  up).  Since  the  enzyme  is  only  adapted  to  unite  with 
certain  stereo-isomers  of  the  amino-acids  only  these  stereo- 
isomers  can  be  transported  into  the  structure  which  the  enzyme 
is  engaged  in  building  up.  In  other  words,  the  stereo-isomeric 
structure  of  the  naturally  occurring  proteolytic  enzymes  neces- 
sarily determines  the  stereo-isomeric  structure  of  all  of  the  natu- 
rally occurring  proteins,  and  for  the  same  reason  that  these  enzymes 
can  only  split  certain  amino-acid  unions,  they  can  only  cause 
these  unions  to  arise.  Hence  the  occurrence  of  only  those  stereo- 
isomeric  groups  within  the  protein  molecule  which  are  open  to 
attack  by  the  proteolytic  enzymes  may  be  regarded  as  a  conse- 
quence of  their  power  to  react  with  the  digestive  enzymes  (17). 

From  the  above-cited  results  of  Fischer  and  Abderhalden  it 
is  evident  that  not  only  the  stereo-isomeric  structure  of  the 


374  CHEMICAL  DYNAMICS 

amino-acid  radicals  in  a  polypeptid  is  of  great  importance  in 
determining  the  susceptibility  of  the  polypeptids  to  hydrolyis 
by  trypsin,  but  also  the  order  in  which  the  amino-acid  groups 
are  combined  and  the  nature  of  these  groups  themselves,  apart 
altogether  from  their  optical  configuration.  Thus  alanyl-glycin, 
CH3.CH2NH2.CO.NH.CH2.COOH,  is  readily  hydrolysed;  while 
the  isomeric  glycyl-alanin,  NH2.CH2.CO.NH.CH.CH3.COOH,  is 
not.  When  alanin  is  the  acyl  radical,  as  in  alanyl-glycin,  alanyl- 
alanin  and  alanyl-leucin,  hydrolysis  occurs,  but  it  does  not  when 
leucin,  valin  or  aminobutyric  acid  are  the  acyl  radicals  (for  in- 
stance, leucyl-alanin,  leucyl-glycin  and  leucyl-prolin). 

The  number  of  amino-acid  groups  within  the  molecule  is  also 
of  great  importance.  Thus  tetraglycyl-glycin  is  hydrolysed  in 
the  presence  of  trypsin,  while  glycyl-glycin,  diglycyl-glycin  and 
triglycyl-glycin  are  not;  other  things  being  equal  it  is  evident 
that  mere  length  of  the  peptid  chain  per  se  confers  upon  it  greater 
susceptibility  to  attack  by  this  enzyme. 

It  will  be  observed,  on  surveying  the  above  results,  that  a 
number  of  polypeptids  are  not  hydrolysable  by  pure  trypsin 
which  nevertheless  are  built  up  out  of  naturally  occurring  radicals. 
Particularly  resistant  to  hydrolysis  are  those  peptids  which  con- 
tain a  preponderance  of  glycin,  phenylalanin  or  pyrrolidin- 
carboxylic  acid.*  In  the  light  of  the  theory  presented  above, 
therefore,  the  question  forces  itself  upon  us,  how  can  such  peptid 
groups  come  to  form,  as  they  do,  constituent  parts  of  the  mole- 
cules of  many  proteins?  Obviously,  if  they  cannot  be  digested 
by  trypsin  and  our  theory  is  valid  then  they  cannot  have  been 
introduced  into  the  protein  molecule  through  the  agency  of  trypsin ; 
they  must  have  been  introduced  through  the  agency  of  some 
other  enzyme  or  enzymes. 

In  this  connection  the  very  significant  fact  will  be  observed 
that  whereas  Fischer  and  Bergell  succeeded  in  hydrolysing  leucyl- 
alanin  through  the  agency  of  pancreas-extract,  Fischer  and  Abder- 
halden  were  unable  to  secure  the  hydrolysis  of  this  substance 
through  the  agency  of  pure  trypsin.  This  pointed  to  the  exist- 
ence in  the  extract  of  enzymes  other  than  trypsin  which,  unlike 

*  Emil  Fischer  and  E.  Abderhalden  (10).  The  connection  between  these 
results  and  the  theory  of  Kuhne  that  the  proteins  are  built  up  of  hemi-  and 
anti-groups,  respectively,  hydrolysable  and  non-hydrolysable  by  pepsin  and 
trypsin  is  sufficiently  evident  to  require  no  further  comment  here. 


HYDROLYSIS  OF  POLYPEPTIDS  375 

trypsin,  are  able  to  bring  about  the  hydrolysis  of  leucyl-alanm. 
The  possibility  was  thus  clearly  indicated  that  a  variety  of  enr 
zymes  might  be  found  to  exist  in  the  various  tissues  and  tissue 
fluids,  distinguishable  from  one  another  in  then:  power  to  hydro- 
lyse  various  polypeptids,  but  only  with  difficulty  distinguishable 
from  one  another  in  their  action  upon  proteins,  since  every  pro- 
tein, it  might  be  anticipated,  contains  unions  susceptible  to  attack 
by  any  one  of  these  enzymes. 

The  possibility  thus  indicated  has  been  rendered  a  certainty, 
thanks  to  the  extensive  researches  of  Abderhalden  and  his  col- 
laborators (2).  These  investigators  have  employed  the  extracts 
and  press-juices  of  various  organs,  prepared  by  Buchner's  method 
of  grinding  up  with  sand  and  kieselguhr  and  pressing  out  at  a 
pressure  of  100  to  300  atmospheres,  which  contain  a  number  of 
different  enzymes  capable  of  bringing  about  the  hydrolysis  of 
various  polypeptids.  These  enzymes  are  not  so  selective  in  their 
action  as  pure  trypsin,  they  can  hydrolyse  polypeptids  which 
trypsin  cannot,  and  the  press-juices  of  different  organs  exhibit 
characteristic  differences  in  regard  to  the  polypeptids  which 
they  can  and  those  which  they  cannot  attack.  Most  striking 
differences  were  observed  between  the  enzymatic  activities  of 
the  red  blood-corpuscles  and  those  of  the  plasma  which  bathes 
them.  The  red  corpuscles  of  the  horse  (but  not  those  of  the  ox) 
hydrolyse  glycyl-1-tyrosin,  which  is  not  hydrolysed  by  white 
corpuscles  nor  by  plasma  or  serum;  they  also  hydrolyse  diglycyl- 
glycin  which,  it  will  be  recollected,  is  not  hydrolysed  by  trypsin. 
Plasma  and  serum  both  hydrolyse  d-1-alanyl-glycin,  diglycyl 
glycin  and  triglycyl  glycin;  hence  the  enzymatic  activity  of  the 
serum  is  not  attributable  to  trypsin  or  erepsin  absorbed  from 
the  intestinal  wall. 

Pure  pepsin,  prepared  by  Pavlov,  does  not  act  upon  any  of 
the  polypeptids,  whereas  it  splits  the  natural  proteins  into  some 
half-dozen  peptones  and  proteoses,  although  it  does  not  split 
these  substances  (which  are  in  reality  polypeptids)  any  further, 
and  hence  yields  no  amino-acids.  Of  this  fact  two  alternative 
explanations  are  offered  (16).  Either  the  proteins  contain  cer- 
tain types  of  union  (such  as  ether-like  combinations  and  so  forth) 
which  are  not  present  in  the  synthetic  polypeptids  and  which 
are  the  only  points  of  union  which  pepsin  can  attack;  or  else 
the  great  length  of  the  amino-acid  chain  in  the  proteins  confers 


376  CHEMICAL  DYNAMICS 

upon  it  greater  susceptibility  to  pepsin  than  the  shorter  poly- 
peptid  chains  possess.  In  support  of  the  former  view  it  is  pointed 
out  that  although  erepsin,  obtained  from  the  succus  entericus, 
is  able  to  hydrolyse  peptones  and  polypeptids,  yet  it  cannot 
hydrolyse  proteins,  a  fact  which  would  appear  to  point  towards 
the  existence  of  a  few  linkages  in  proteins  which  are  not  pres- 
ent in  polypeptids.  In  support  of  the  latter  view  it  is  pointed 
out  that  polypeptid-chains  of  greater  length  are  more  susceptible 
to  attack  by  other  enzymes  (i.e.,  trypsin)  than  the  shorter  chains, 
so  that  the  possibility  cannot  be  overlooked  that  the  suscepti- 
bility of  proteins  to  attack  by  pepsin  may  merely  be  attributable 
to  their  great  complexity,  i.e.,  to  the  extreme  length  of  the 
polypeptid-chain. 

2.  The  Kinetics  of  the  Hydrolysis  of  Polypeptids  by  Proteo- 
lytic  Enzymes.  —  The  progressive  hydrolysis  of  diglycyl-tyrosin 
in  the  presence  of  trypsin  was  followed  by  Taylor  (19).  He 
states  that  the  results  which  he  obtained  were  irregular  and  un- 
satisfactory, but  he  regarded  these  irregularities  as  being  attrib- 
utable to  analytical  errors. 

The  optically  active  dipeptids,  d-alanyl-d-alanin,  d-alanyl- 
1-leucin  and  glycyl-1-tyrosin,  have  been  employed  by  Abderhalden 
and  Koelker,  Abderhalden  and  Michaelis  and  Abderhalden  and 
Gigon  (3)  (4)  (6)  (14)  in  investigating  the  time  relations  of  their 
hydrolysis  by  trypsin.  The  degree  of  hydrolysis  at  any  moment 
can  readily  be  followed  by  observing  the  optical  rotation  of  the 
solution,  the  rotation  due  to  d-alanyl-d-alanin,  for  example,  being 
negative  (aD20°  =  —21.2°),  while  that  of  the  products  of  complete 
hydrolysis  is  positive.  The  change  in  the  optical  rotation  is,  of 
course,  directly  proportional  to  the  degree  of  hydrolysis.* 

The  relation  between  the  time  and  the  extent  of  hydrolysis 
is,  as  Abderhalden  and  Michaelis  have  shown,  susceptible  of 
fairly  simple  formulation.  The  form  of  relation  which  is  char- 
acteristic for  a  monomolecular  reaction  (that  is,  a  transformation 
which  involves  only  one  species  of  molecule)  is  expressed  by  the 
differential  equation: 


*  Subject  to  a  slight  correction  due  to  the  fact  that  the  specific  rotatory 
powers  of  the  dipeptids  are  not  absolutely  independent  of  their  concentrations. 
Cf.  Koelker  (14). 


KINETICS  OF  HYDROLYSIS 


377 


in  which  x  is  the  amount  transformed  after  time  t,  a  is  the  initial 
quantity  of  substrate  and  k  is  the  velocity-constant  of  the  reaction. 
When  integrated  this  yields  the  equation: 


log 


a  —  x 


=  kt. 


G) 


In  the  derivation  of  this  equation  it  is  to  be  noted  that  the 
velocity  of  the  reverse  reaction  is  regarded  as  being  negligible, 
so  that  no  station  of  equilibrium  is  reached  until  the  transfor- 
mation is  practically  complete.  This  condition  is  tolerably  well 
fulfilled  in  the  reaction  under  consideration,  for  the  hydrolysis 
of  the  polypeptid  does  not  cease  until  the  quantity  of  unde- 
composed  polypeptid  is  practically  unappreciable.  In  the  fol- 
lowing table  are  given  the  values  of  the  constant  k  for  the 
hydrolysis  of  d-alanyl-d-alanin  by  liver  extract  at  37  degrees 
calculated  by  Abderhalden  and  Michaelis  from  the  experimental 
values  of  a  —  x  and  t  obtained  by  Abderhalden  and  Koelker. 
In  every  case  the  initial  concentration  of  the  dipeptid  was  such 
that  1.45  grams  of  the  substance  were  dissolved  in  6  cc.  of  the 
digest. 


Time 
in 
min- 
utes 

1.45  units 
of  dipeptid 
+  6  cc*  of 
ferment  so- 
lution k  = 

Time 
in 
min- 
utes 

1.45  units 
of  dipeptid 
-f-  4  cc.  of 
ferment  solu- 
tion k  = 

Time 
in 
min- 
utes 

1.45  units 
of  dipeptid 
+  3  cc.  of  fer- 
ment solution 
*  = 

Time  • 
in 
min- 
utes 

1.45  units 
of  dipeptid 
+  2  cc.  of  fer- 
ment solution 
fc- 

3 

0.0453 

3 

0.0192 

5 

0.0125 

5 

0.00710 

7 

0.0390 

7 

0.0183 

6.5 

0.0132 

11 

0.00847 

11 

0.0342 

10 

0.0202 

7.5 

0.0134 

15 

0.00834 

13 

0.0336 

11 

0.0197 

16 

0.0142 

23 

0.00901 

18 

0.0380 

16 

0.0214 

22 

0.0161 

31 

0.01020 

20 

0.0373 

17 

0.0203 

23 

0.0163 

41 

0.01216 

24 

0.0399 

25 

0.0268 

28 

0.0192 

53 

0.01474 

27 

0.0451 

30 

0.0294 

29 

0.0196 

65 

0.01920 

34 

0.0430 

34 

0.0342 

30 

0.0209 

80 

0.01810 

35 

0.0359 

38 

0  0232 

45 

0.0265 

It  is  evident  that,  within  the  experimental  error,  k  calculated  from 
formula  (i)  is  tolerably  constant  for  the  higher  concentrations  of 
ferment  solution  (6  cc.  of  ferment  solution);  for  lower  ferment- 
concentrations,  however,  the  value  of  k  does  not  even  approxi- 
mate to  constancy,  but  rises  markedly  as  hydrolysis  proceeds; 
in  other  words  the  velocity  of  the  transformation  does  not  fall 


378 


CHEMICAL  DYNAMICS 


off  so  rapidly  as  it  should  were  it  a  simple  monomolecular  re- 
action of  which  the  velocity  constant  is  simply  enhanced  in 
magnitude  by  the  presence  of  the  ferment. 

In  the  derivation  of  equation  (i)  it  is  assumed  that  the  velocity 
of  transformation  at  any  moment  is  proportional  to  the  mass  of 
substrate  which  is  at  that  moment  undergoing  change.  The 
theoretical  velocity  therefore  falls  off  in  direct  proportion  as  the 
substrate  is  used  up.  The  next  simplest  assumption,  taking 
cognizance  of  the  fact  that  the  velocity  of  transformation  does 
not  decrease  with  the  rapidity  demanded  by  the  monomolecular 
formula,  is  that  the  velocity  of  transformation  is  independent 
of  the  mass  of  the  substrate,  and  is  therefore  expressed  by  the 
equation  : 

dx 


which,  when  integrated,  yields 

x  =  kit.  (ii) 

Calculating  the  values  of  ki  from  this  formula,  for  the  above 
four  series  of  experimental  data  Abderhalden  and  Michaelis 
obtained  the  following  results  : 


1  .  45  units  of 

1.45  units  of 

1  .  45  units  of 

1.45  units  of 

dipeptid  +  6  cc. 
of  ferment  solution 

dipeptid  +  4  cc. 
of  ferment  solution 

dipeptid  +  3  cc. 
of  ferment  solution 

dipeptid  +  2  cc. 
of  ferment  solution 

0.1300 

0.0600 

0.0380 

0.0240 

0.0843 

0.0529 

0.0400 

0.0255 

0.0713 

0.0540 

0.0400 

0.0240 

0.0708 

0.0518 

0.0369 

0.0239 

0.0639 

0.0494 

0.0368 

0.0242 

0.0595 

0.0494 

0.0365 

0.0241 

0.0538 

0.0456 

0.0368 

0.0228 

0.0504 

0.0420 

0.0366 

0.0212 

0.0411 

0.0397 

0.0314 

0.0180 

0  0332 

0  0302 

It  is  evident  that  k1}  calculated  from  formula  (ii),  tends  to 
approximate  constancy  for  the  lower  ferment  concentrations  (2  cc. 
and  3  cc.),  while  it  altogether  fails  to  do  so  for  the  higher  ferment- 
concentrations.  To  the  latter  solutions,  as  we  have  seen,  equa- 
tion (i)  applies  tolerably  well.  Neither  equation,  therefore, 
taken  by  itself,  represents  the  entire  process;  each  applies  under 


KINETICS  OF  HYDROLYSIS 


379 


certain  limiting  conditions.  The  possibility  was  thus  indicated 
that  a  combination  of  the  two  formulae  might  be  found  to  ade- 
quately represent  all  of  the  phenomena.  Obviously  the  process 
represented  by  equation  (i)  plays  a  predominant  part  when  the 
ferment-concentration  is  high;  that  represented  by  equation 
(ii)  plays  a  predominant  part  when  the  ferment-concentration 
is  low.  We  must  therefore  introduce  into  the  combined  equation 
some  factor  e  which  will  denote  the  proportionality  between 
the  two  processes,  and  which  will  be  dependent  upon  the  mass 
of  ferment.  We  thus  obtain  the  equation : 


lognat. 


a  —  x 


ex 


kzt; 


(iii) 


introducing  the  modulus  of  the  natural  logarithms  (0.4343)  we 
obtain 

a 


logic 


a  —  x 


which  may  be  written 
logi 


+  0.4343  ex  =  0.4343  fcrf, 


+  0.4343  ex  = 


a  —  x 


(iv) 


The  following  are  the  values  of  kz  calculated  by  Abderhalden 
and  Michaelis  from  equation  (iv)  for  the  four  series  of  deter- 
minations cited  above: 


1.45  units  of 

1  .  45  units  of 

1  .  45  units  of 

1.45  units  of 

dipeptid  +  6  cc. 
of  ferment  solution 

dipeptid  +  4  cc. 
of  ferment  solution 

dipeptid  +  3  cc. 
of  ferment  solution 

dipeptid  +  2  cc. 
of  ferment  solution 

e  =  0.7 

e  =  1.5 

6  =  3.0 

e  =  10.0 

100  kt  = 

100*3  = 

100A3  = 

100  kt  = 

(8.49) 

5.74 

6.20 

11.1 

6.63 

6.52 

6.54 

11.9 

5.49 

5.90 

6.56 

11.2 

5.52 

5.51 

6.22 

11.3 

5.68 

5.25 

6.41 

11.5 

5.54 

5.91 

6.40 

11.7 

5.63 

5.91 

6.72 

11.1 

6.04 

6.15 

6.53 

11.1 

5.55 

6.17 

6.17 

(9.6) 

6.64 

6.58 

With  the  exception  of  the  two  values  enclosed  in  brackets, 
which  are  obviously  influenced  by  experimental  errors,  the  value 
of  &3  for  each  set  of  determinations  is  evidently,  within  the  ex- 


380  CHEMICAL  DYNAMICS 

perimental  error,  constant.  The  value  of  e,  it  is  evident,  rises 
with  decreasing  ferment-concentration,  but  not  in  direct  pro- 
portion. 

It  is  of  great  interest  to  observe  that  equation  (iii)  and  (iv) 
is  of  the  same  form  as  that  which  Henri  (13)  (6)  has  found  to 
hold  good  for  the  inversion  of  cane  sugar  by  invertase.  The 
theoretical  foundation  of  Henri's  equation,  therefore,  calls  for 
consideration  in  this  connection.* 

Henri  started  from  the  point  of  view  that  the  ferment,  in  the 
presence  of  a  substrate  which  is  undergoing  digestion,  may  con- 
ceivably exist  in  three  modifications,  namely,  in  combination 
with  the  substrate  (concentration  =  F^,  in  combination  with 
the  products  (concentration  =  Fp),  and  in  the  free  condition 
(concentration  =  Ff).  Calling  the  total  concentration  of  ferment 
F,  we  obviously  have: 

F  =  F.  +  F9  +  Ff.'  (v) 

Assuming  that  in  the  formation  of  the  ferment-substrate 
compound  one  molecule  of  ferment  unites  with  one  molecule 
of  substrate  we  have,  from  the  mass-law: 

F,(a-x)=±F8,  (vi) 

in  which  —  is  the  equilibrium-constant  of  the  reaction. 

Similarly  assuming  that  in  the  formation  of  the  ferment- 
products  compound  one  molecule  of  ferment  unites  with  one 
molecule  of  the  hydrolysis-products  we  have: 

F>*  =  l~Ff,  (vii) 

in  which  -  is  the  equilibrium-constant  of  the  reaction. 
n 


Combining  equations  (v),  (vi)  and  (vii)  we  obtain: 

F 


(viii) 


l  +  m(a-x)+nx 
and 

j?  mF  (fl  ~  *)  f^\ 

'•"  ' 


*  The  derivation  of  Henri's  equation  which  follows  is,  essentially,  quoted 
from  Taylor  (19). 


KINETICS  OF  HYDROLYSIS  381 

According  to  Henri,  the  velocity  of  hydrolysis  might  con- 
ceivably be  directly  proportional  to  the  mass  of  the  ferment- 
substrate  compound  (=  Fa)  or  to  the  mass  of  the  free  ferment 
and  that  of  the  substrate.  In  the  former  case  we  obtain: 

dx  kmF(a  —  x)  .  . 

~j~4  ~  i — i    _  („ \ — i '  W 

at      1  ~r  m  (a  —  x )  -r  nx 

in  which  k  is  the  velocity-constant  of  the  reaction.  In  the  latter 
case  we  have: 

dx  =         kF(a  -  x)  (  .. 

dt       1  +  m  (a  —  x)  -f  nx 

Obviously  both  equations  are  identical  in  form;  the  former 
when  integrated,  leads  to  the  equation: 

(1  +  na)  log h  (m  —  n)  x  =  kjt,  (xii) 

a  ~~  x 

in  which  ks  is  a  constant  which  is  directly  proportional  to  the 
total  concentration  of  ferment,  and  n  and  m  are  independent 
of  the  mass  of  ferment  or  substrate,  but  are  obviously  dependent 
upon  the  temperature  and  the  conditions  (reaction,  etc.)  of  the 
experiment.  Putting 


1  +na 

we  obviously  regain  the  equation  of  Abderhalden  and  Michaelis. 
The  condition  that  m  and  n  should  be  independent  of  the  mass 
of  ferment,  however,  is  obviously  not  fulfilled  by  Abderhalden 
and  Michaelis'  results,  since  €  varies  notably  with  the  concen- 
tration of  the  ferment;  while  ks  does  not  approximate  to  direct 
proportionality  to  the  mass  of  ferment. 

In  the  derivation  of  Henri's  equation  it  will  be  evident  that ' 
many  simplifying  assumptions  are  made  which  are  not  justified 
by  anything  save  the  fact  that  they  afford  the  simplest  con- 
ception of  the  relations.  Thus  it  is  assumed  that  the  active 
mass  of  the  ferment  in  so  far  as  hydrolysis  is  concerned  is  directly 
proportional  to  its  actual  mass,  that  one  molecule  of  ferment 
reacts  with  one  molecule  of  substrate,  that  one  molecule  of  fer- 
ment reacts  with  one  molecule  of  the  products  of  hydrolysis, 
that  the  concentration  of  the  ferment-substrate  compound  is 


382 


CHEMICAL  DYNAMICS 


evanescent  in  comparison  with  that  of  the  substrate  and  so  forth. 
If  we  try  to  generalize  Henri's  equation  by  removing  these 
simplifying  assumptions  we  obtain  equations  which  are,  mathe- 
matically speaking,  unmanageable.  The  surprising  thing  is, 
therefore,  not  that  Henri's  equation  fails  to  adequately  repre- 
sent Abderhalden  and  Koelker's  results,  but  that,  if  the  con- 
ditions in  the  system  are  as  complex  as  those  which  Henri  depicts, 
any  simple  relation  can  be  found  which  will  express  the  progress 
of  the  reaction  with  any  approach  to  fidelity. 

Computing  the  average  values  of  100  &3  calculated  from  the 
equation  (iv)  for  the  various  ferment-concentrations  employed 
by  Abderhalden  and  Koelker  we  obtain: 


cc.  of  ferment  solu- 
tion in  6  cc.  of  digest 

100*3 

cc.  of  ferment  solu- 
tion in  6  cc.  of  digest 

lOOifca 

2 
3 

11.6 

6.45 

4 
6 

6.01 
5.76 

from  which  it  is  evident  that  the  velocity-constant  of  hydrolysis, 
when  computed  from  Henri's  equation,  far  from  being  directly 
proportional  to  the  ferment-mass  is  actually  greater  the  more 
dilute  the  ferment.  It  appears  distinctly  within  the  bounds  of 
possibility  that  the  intensity  of  the  hydrolysing  activity  of  the 
ferment  is  relatively  greater  the  more  dilute  its  solution.  This, 
in  turn,  suggests  the  possibility  that  the  ferment  may  exist  both 
in  an  active  and  inactive  form  (active  and  inactive,  that  is,  with 
respect  to  the  acceleration  of  hydrolysis).  The  proportion  of  in- 
active ferment  would,  of  course,  depend,  not  only  upon  the  total 
mass  of  ferment  in  the  system,  but  upon  the  mass  of  uncombined 
active  ferment.  If  this  were  the  case  then  k  and  e  in  Abder- 
halden and  Michaelis'  equation,  accepting  Henri's  derivation  of 
the  equation,  might  be  expected  to  be  functions,  not  only  of  the 
total  ferment  mass  and  of  the  relation  between  the  mass  of  free 
active  ferment  and  that  of  the  substrate  and  products  respec- 
tively, but  also  of  the  relation  between  the  proportion  of  ferment 
in  the  inactive  condition  and  the  mass  of  the  uncombined  active 
ferment  (Cf.  Chap.  XVII). 

On  the  other  hand,  it  is  possible  that  the  coincidence  between 
Abderhalden  and  Michaelis'  equation  and  that  of  Henri  is  merely 
formal,  that,  as  Abderhalden  and  Michaelis  suggest,  the  true 


KINETICS  OF  HYDROLYSIS 


383 


equation  expressing  the  course  of  hydrolysis  is  in  reality  equation 
(i),  but  that  as  hydrolysis  proceeds  the  reaction  is  subject  to  an 
acceleration,  possibly  attributable,  either  directly  or  indirectly, 
to  the  products  of  the  reaction. 

Euler  (9)  has  studied  the  hydrolysis  of  glycin  by  erepsin, 
obtained  from  the  intestinal  wall.  The  method  employed  was 
that  of  following  the  hydrolysis  by  measuring  the  progressive 
alteration  of  the  electrical  conductivity  of  the  solution,  direct 
proportionality  between  the  alteration  in  conductivity  and  that 
of  the  dipeptid-concentration  having  previously  been  established. 
He  finds  that  the  velocity  of  hydrolysis  is  very  intimately  de- 
pendent upon  the  alkalinity  of  the  solution,  thus: 

1/10  N  GLYCYL-GLYCIN.    5  G.  EREPSIN  [POWDER  IN  100  CC. 

Alkali-concentration  N 0        0.04    0.05    0.075    0.10 

Reaction-velocity  constant  X  1000    0.05    7.0      6.2      2.6       0.2 

For  a  given  initial  NaOH,  dipeptid  and  erepsin  concentration 
the  monomolecular  formula  represents  the  progress  of  this  re- 
action with  tolerable  fidelity,  as  the  following  results  show: 

0.10 N  GLYCYL  GLYCIN.    5  GRAMS  EREPSIN  IN  100  CC. 


0.04  AT  Na 


Minutes 

1000  (a  -  x) 

1000  k 

Minutes 

1000  (a  -  z) 

1000  k 

o 

920 

o 

935 

7 

819 

7.20 

10 

806 

6.46 

15 

721 

7.08 

17 

739 

6.00 

22 

649 

6.88 

25 

654 

6.18 

30 

579 

6.70 

30 

622 

6.90 

0.05  N  Na 


Within  certain  limits  the  reaction-velocity  is  independent  of 
the  initial  concentration  of  this  dipeptid,  but  this  holds  good 
only  within  certain  limits  of  the  proportion  enzyme  :  substrate. 
If  the  enzyme-concentration  is  small,  for  a  given  Na-concentra- 
tion  the  reaction-velocity  rises  with  increasing  concentration  of 
glycyl-glycin.  Euler  attributes  this  fact  to  neutralization  of 
injurious  excess  of  NaOH  by  the  additional  glycyl-glycin.  The 
progressive  change  in  the  reaction-constant  as  hydrolysis  proceeds 
may  doubtless  be  attributed  to  the  fact  that  the  substrate  and 
products  are  possessed  of  different  combining  capacities  for  bases, 
so  that  the  proportion  of  free  base  alters  as  the  reaction  proceeds. 


384 


CHEMICAL  DYNAMICS 


Euler  has  arrived  at  the  conclusion  that  in  solutions  of  glycyl- 
glycin  which  contain  a  base,  only  the  salt  of  the  dipeptid  under- 
goes hydrolysis.  In  terms  of  recent  theories  of  catalysis  (18) 
this  may  be  held  to  indicate  that  the  substance  which  actually 
undergoes  hydrolysis  is  the  dipeptid  ion. 

For  high  ferment-concentrations  the  velocity  of  the  hydrolysis 
of  glycyl-glycin  is,  according  to  Euler,  directly  proportional  to 
the  concentration  of  erepsin.  If,  however,  the  ferment  concen- 
tration is  low,  then  the  value  of  k,  calculated  from  the  mono- 
molecular  formula,  increases  much  more  rapidly  than  the  con- 
centration of  erepsin. 

The  influence  of  various  added  substances  (salts,  ammo-acids, 
and  so  forth)  upon  the  rate  of  hydrolysis  of  dipeptids  by  liver- 
extract  has  been  studied  by  Abderhalden  and  Gigon  (3).  They 
find  that  dilute  solutions  of  KCN  accelerate  while  strong  solu- 
tions greatly  retard  the  hydrolysis  of  d-1-leucyl-glycin;  sodium 
fluoride  strongly  retards  the  hydrolysis  of  d-1-leucyl-glycin  and 
of  glycyl-1-tyrosin;  "physiological  salt  solution"  has  no  effect 
upon  the  rate  of  hydrolysis.  In  high  concentrations  both  mag- 
nesium chloride  and  sulphate  depress  the  rate  of  hydrolysis; 
calcium  chloride  accelerates  the  hydrolysis  and  strontium  chlo- 
ride is  indifferent,  the  substrates  being  d-1-leucyl-leucin  and  glycyl- 
1-tyrosin. 

The  effect  of  amino-acids  upon  the  rate  of  hydrolysis  is  of 
surpassing  interest  since  these  are  the  products  of  the  reaction, 
and  if  they  exert  an  influence  upon  the  rate  of  hydrolysis  they  will 
induce  more  or  less  marked  deviations  in  the  relation  between 
the  duration  and  the  extent  of  hydrolysis  from  that  which  would 
be  indicated  by  the  monomolecular  law  or  by  Henri's  modifi- 
cation of  the  monomolecular  law,  described  above.  Employing 
glycyl-1-tyrosin  they  find  that : 


The  hydrolysis  is 
accelerated  by 

The  hydrolysis  is 
unaffected  by 

The  hydrolysis  is 
slightly  retarded  by 

The  hydrolysis  is 
strongly  retarded  by 

1-alanin 

glycocoll 
d-leucin 

d-1-alanin 
d-valin 

d-alanin 
d-1-valin 

d-1-leucin 

1-leucin 

d-1-serin 

1-serin 

1-tyrosin 

d-isoserin 

d-1-isoserin 

1-isoserin 

ORDER  IN  WHICH  AMINO-ACIDS  ARE  SPLIT          385 

In  addition  it  was  observed  that  d-glutamic  acid,  d-tryptophane, 
1-diamino-trioxydodecanic  acid,  d-1-aminobutyric  acid  and  phenyl- 
alanin  depress  the  rate  of  hydrolysis. 

In  every  case  it  will  be  observed  that  the  effects  of  optical 
antipodes  are  considerably  different,  in  quantity  or  even,  in  the 
case  of  alanin,  in  sense,  from  one  another  and  that  the  racemic 
body  affects  the  hydrolysis  in  an  intermediate  manner.  Of 
great  significance  is  the  fact  that  1-tyrosin,  which  is  itself  a  prod- 
uct of  the  hydrolysis  of  glycyl-1-tyrosin,  strongly  retards  the 
hydrolysis,  although  glycocoll,  which  is  the  other  product,  does 
not.  Abderhalden  and  Gigon  attribute  this  to  a  binding  of  the 
ferment  by  the  tyrosin.  The  affinity  between  glycocoll  and  the 
ferment  is  slight  and  so  this  product  does  not  retard  hydrolysis 
so  markedly.  It  is  possible  that  the  low  degree  of  affinity  be- 
tween glycocoll  and  the  majority  of  the  proteolytic  ferments  is 
responsible  for  the  difficultly  digestible  character  of  many  of  the 
peptids  which  are  rich  in  glycocoll. 

The  influence  of  temperature  upon  the  rate  of  hydrolysis  of 
glycyl-1-tyrosin  and  d-1-leucyl-glycin  by  liver  and  pancreas- 
extracts  has  also  been  studied  by  Abderhalden,  Caemmerer  and 
Pincussohn  (2).  In  each  case,  within  certain  limits,  the  rate  of 
hydrolysis  is  very  greatly  accelerated  by  a  rise  in  temperature. 
Very  high  temperatures,  naturally,  delay  or  prevent  hydrolysis 
by  destruction  of  the  enzyme.  For  liver  extract,  working  upon 
these  substrates,  the  temperature-optimum  proved  to  be  about 
55  degrees;  for  pancreas-extract  between  45  and  50  degrees. 

3.  The  Order  in  which  Amino-acids  are  Split  off  from  Poly- 
peptids  by  Proteolytic  Ferments.  —  The  optical  properties  of 
certain  polypeptids  and  of  their  possible  decomposition-products 
have  been  utilized  by  Abderhalden  and  Koelker  (4),  Abderhalden 
and  Brahm  (1)  and  Abderhalden,  Koelker  and  Medigreceanu  (5) 
in  the  attempt  to  ascertain  which  point  in  a  tri-  or  tetra-peptid 
is  first  attacked  by  a  proteolytic  ferment.  Thus  1-leucyl-glycyl- 
d-alanin  has  a  molecular  rotation  *  of  +52  degrees.  It  might 
conceivably  yield  in  the  first  place  either  1-leucyl-glycin  (+172 
degrees)  and  d-alanin  (+2  degrees),  or  else  glycyl-d-alanin  (—73 
degrees)  and  1-leucyl  (  —  13  degrees),  or  else,  through  simultaneous 
splitting  of  both  — NHOC—  bonds,  all  three  of  the  products 

*  The  rotations  given  are  1/100  of  the  molecular  rotations.  Cf.  Koelker 
(14). 


386  CHEMICAL  DYNAMICS 

1-leucyl  (—13  degrees),  glycin  (0  degrees)  and  d-alanin  (+2 
degrees).  If  the  first  link  to  be  broken,  therefore,  is  that  be- 
tween the  glycyl  and  alanyl  groups  the  positive  rotation  of  the 
original  solution  should  increase  until  it  reaches  a  maximum. 
If  this  link  were  the  only  one  attacked  then  this  maximum  rota- 
tion should  be  that  of  a  mixture  of  1-leucyl-glycin  and  d-alanin, 
i.e.,  over  three  times  that  of  the  original  solution.  If  the  first 
link  to  be  broken  were  that  between  the  leucyl-  and  the  glycyl- 
groups  the  original  positive  rotation  should  decrease  and  finally 
become  negative.  If  the  two  links  were  split  simultaneously 
the  positive  rotations  should  decrease  until  it  became  very  slightly 
negative.  These  possibilities  are  made  clear  by  the  following 
diagram: 


1-leueyl-glycyl-d-alanin 
0° 


+  172 


-73 


The  experimental  result,  employing  trypsin,  is  that  the  rota- 
tion at  first  increases  to  an  extent  of  about  40  per  cent.  Hence 
1-leucyl-glycin  must  be  liberated  and  the  point  of  first  attack 
(or,  at  least,  most  rapid  attack)  must  be  the  bond  between  the 
glycyl  and  the  alanyl  groups.  After  attaining  a  maximum 
positive  rotation  only  40  per  cent  in  excess  of  that  of  the  original 
solution,  however,  the  rotation  declines  again,  owing  to  the 
hydrolysis  of  the  1-leucyl-glycin.  Hence  the  enzyme  does  not 
complete  the  decomposition  of  the  one  bond  before  attacking 
the  other.  In  other  words  the  two  reactions  proceed  side  by 
side  but  at  different  velocities,  that  of  the  splitting  of  the  glycyl- 
alaniri  bond  being  the  most  rapid.  The  leucyl-glycin  bond, 
however,  does  not  appear  to  be  attacked  at  all  while  it  is  bound 
up  in  the  tripeptid  molecule,  there  being  no  evidence  of  the 
formation  of  glycyl-d-alanin.  The  progress  of  the  reaction  is 
therefore,  it  would  appear,  the  following,  first  the  glycyl-alanin 
link  is  broken  and  1-leucyl-glycin  and  alanin  are  split  off.  The 
v.elocity  of  this  reaction  is  so  much  greater  than  that  of  the  other 
possible  reactions  (splitting  of  the  leucyl-glycyl  link)  that  the 
latter  reaction  does  not  occur  to  any  appreciable  extent.  The 
1-leucyl-glycin  which  is  thus  formed  is  immediately  attacked  by 


ORDER  IN  WHICH  AMINO-ACIDS  ARE  SPLIT          387 

the  ferment.  The  substrate  of  this  second  reaction  being,  however, 
a  product  of  the  first,  its  velocity  is  at  first  low,  since  the  sub- 
strate concentration  is  low;  but  as  velocity  of  the  first  reaction 
declines,  owing  to  the  consumption  of  the  substrate,  that  of 
the  second  reaction  increases,  owing  to  the  increase  in  the  mass 
of  its  substrate. 

When  however,  instead  of  trypsin,  yeast  endotryptase  is  em- 
ployed as  the  ferment,  not  the  glycyl-alanin  bond  but  the  leucyl- 
glycyl  bond  is  first  attacked.  There  is  thus  no  question  but 
that  the  mode  of  action  of  yeast  endotryptase  is  quite  different 
from  that  of  trypsin  and  a  means  of  sharply  distinguishing  be- 
tween different  enzymes  is  clearly  indicated.  It  will  also  be 
evident  that  in  digests  containing  a  mixture  of  proteolytic  enyzmes, 
such  as  occurs  in  tissue-extracts,  etc.,  the  conditions  of  hydrolysis 
must  be  much  more  complex  than  they  are  in  digests  containing 
only  a  single  enzyme;  since  under  such  circumstances  two  or 
more  parallel  reactions  may  be  occurring,  possessed  of  different 
specific  velocities,  at  each  step  in  the  progressive  hydrolysis  of 
of  the  peptid  or  protein. 

Glycyl-d-alanyl-glycin  is  first  attacked  by  trypsin  at  the  glycyl- 
d-alanyl  junction,  by  yeast  endotryptase  at  the  alanyl-glycin 
junction;  d-alanyl-glycyl-glycyl-glycin  is  first  attacked  by  trypsin 
at  the  glycyl-glycin  junction. 

LITERATURE   CITED 

(1)  Abderhalden,  E.,  and  Brahm,  C.,  Zeit.  f.  physiol.  Chem.,  57  (1908), 

p.  342. 

(2)  Abderhalden,  E.,  and  Collaborators  (Babkin,  Bergell,  Bloch,  Damm- 

hahn,  Deetjen,  Gigon,  Heise,  Hunter,  Kautsch,  Koelker,  Lussana, 
Manwaring,  McLester,  Medigreceanu,  Michaelis,  Oppler,  Pilliet, 
Pincussohn,  Pringsheim,  Rona,  Samuely,  Schittenhelm,  Teruuchi, 
Walther,  Weichardt).  Zeit.  f.  physiol.  Chem.,  39  (1903),  p.  9; 
46  (1905),  pp.  176  and  187;  47  (1906),  pp.  159,  346,  359,  391,  466; 
48  (1906),  pp.  537,  557;  49  (1906),  pp.  1,  21,  26,  31;  51  (1907),  pp. 
294,  308,  334;  54  (1908),  p.  363;  55  (1908),  pp.  371,  377,  384,  390, 
395,  416;  57  (1908),  p.  332;  59  (1909),  p.  249;  60  (1909),  p.  415;  61 
(1909),  p.  200;  62  (1909),  pp.  120, 136,  145,  243;  66  (1910),  pp.  265, 
277;  68  (1910),  p.  471. 

(3)  Abderhalden,  E.,  and  Gigon,  A.,  Zeit.  f.  physioL  Chem.,  53  (1907), 

p.  251. 

(4)  Abderhalden,  E.,  and  Koelker,  A.  H.,  Zeit.  f .  physiol.  Chem.,  51  (1907), 

p.  294. 


388  CHEMICAL  DYNAMICS 

(5)  Abderhalden,  E.,  and  Koelker,  A.  H.,  and  Medigreceanu,  F.,  Zeit.  f. 

physiol.  Chem.,  62    1909),  p.  145. 

(6)  Abderhalden,  E.,  and  Michaelis,  L.,  Zeit.  f.  physiol.  Chem.,  52  (1907), 

p.  326. 

(7)  Bayliss,  W.  M.,  "The  Nature  of  Enzyme  Action,"  London,  1908. 
.  (8)   Bredig,  G.,  Ergeb.  d.  Physiol.,  1  Abt.  1  (1902),  p.  134. 

(9)   Euler,  H.,  "Allgemeine  Chemie  der  Enzyme,"  Weisbaden,  1910. 

(10)  Fischer,  E.,  and  Abderhalden,  E.,  Zeit.  f .  physiol.  Chem.,  39  (1903),  p. 

81. 

(11)  Fischer,  E.,  and  Abderhalden,  E.,  Zeit.  f.  physiol.  Chem.,  46  (1905),  p. 

52;  51  (1907),  p  264. 

(12)  Fischer,  E.,  and  Bergell,  P.,  Ber.  d.  d.  chem.  Ges.,  36  (1903),  p.  2592; 

37  (1904),  p.  3103. 

(13)  Henri,  V.,  "Lois  generales  de  Faction  des  Diastases,"  Paris  (1903),  p. 

92. 

(14)  Koelker,  A.  H.,  Journ.  Biol.  Chem.,  8  (1910),  p.  145. 

(15)  Oppenheimer,  C.,  "Ferments  and  their  Actions,"  Erlangen  (1901), 

English  trans.,  London  (1901). 

(16)  Plimmer,  R.  H.  A.,  "The  Chemical  Constitution  of  the  Proteins," 

London  (1908),  Pt.  2,  p.  50. 

(17)  Robertson,  T.  Brailsford,  "The  Proteins,"  Univ.  of  California  Publ. 

Physiol.,  3  (1909),  p.  123. 

(18)  Stieglitz,  J.,  Amer.  Chem.  Journ.,  39  (1908),  p.  29. 

(19)  Taylor,  A.  E.,  "On  Fermentation,"  Univ.  of  California  Publ.  Pathol. 

1  (1907),  p.  87. 

(20)  Vernon,  H.  M.,  " Intracellular  Enzymes,"  London,  1908. 


CHAPTER  XVI 
THE  HYDROLYSIS  OF  THE  PROTEINS 

1.  The  Proteolytic  Enzymes  as  Catalysors.  —  The  fact  that 
proteins,  in  the  presence  of  water,  can  be  hydrolysed  to  ammo- 
acids  by  prolonged  heating,  e.g.,  by  superheated  steam  (126) 
(80)  (81)  (76)  (69)  (70)  (87)  (115)  without  the  addition  to  the 
system  of  any  acid,  alkali  or  ferment,  indicates  that  the  process 
of  hydrolysis  is  occurring,  although  slowly,  at  all  temperatures 
and  in  the  absence  of  catalysors  other  than,  possibly,  the  hydrogen 
or  hydroxyl  ions  of  water  itself.  The  fact  that,  at  ordinary 
temperatures,  catalysors  can  bring  about  the  hydrolysis  of  pro- 
teins shows  that  even  at  these  temperatures  the  proteins  are  not 
in  equilibrium  with  their  products.  The  influence  of  rising  tem- 
perature upon  a  chemical  reaction  is  always  twofold;  it  shifts 
the  station  of  equilibrium  in  one  sense  or  in  the  opposite  and, 
always,  it  accelerates  the  reaction  to  a  greater  or  less  degree 
(i.e.,  magnifies  the  velocity-constant).  The  action  of  heat  upon 
proteins  must  be  in  all  cases  to  shift  the  station  of  equilibrium 
in  the  direction  of  polymerization  (i.e.,  condensation)  since  the 
reaction  of  hydrolysis  is  faintly  exothermic,  but  the  fact  that 
fairly  complete  hydrolysis  occurs  at  temperatures  above  100 
degrees  shows  that  the  shift  in  the  equilibrium  between  the  lower 
protein  complexes  and  the  amino-acids  which  are  the  products  of 
their  hydrolysis  is  not  so  great  as  to  extinguish  the  reaction  of 
hydrolysis.  The  effect  of  high  temperatures  in  accelerating  the 
auto-hydrolysis  of  proteins  is  to  be  looked  upon,  therefore,  as 
that  of  rendering  readily  detectable,  through  acceleration,  a 
reaction  which  occurs,  although  slowly,  at  all  temperatures  (133). 
It  is  possible  to  demonstrate  directly,  however,  and  without 
appeal  to  inference,  that  the  hydrolysis  of  proteins  in  neutral 
watery  solutions  does  occur  at  normal  temperatures;  the  velocity 
of  hydrolysis  is,  however,  usually  very  low  under  these  conditions. 
Taylor  has  shown  that  an  appreciable  proportion  of  pure  sterile 
globulin  kept  in  distilled  water  at  ordinary  temperatures  for 
18  months  is  hydrolysed  to  proteoses;  he  has  also  found  that 

389 


390  CHEMICAL  DYNAMICS 

leucin  may  be  recovered  from  a  sterile  suspension  of  casein  in 
pure  water  and  that  arginin  may  be  recovered  from  a  solution 
of  protamin  sulphate  in  pure  water,  both  after  the  lapse  of  a 
year  or  more  (133);  other  examples  of  the  slow  autohydrolysis 
of  proteins  in  pure  water  might  be  adduced  (133)  (131).  In  a 
few  cases,  however,  the  autohydrolysis  occurs  at  a  readily  meas- 
ureable  velocity.  I  have  found  (110)  that  the  velocity-constant 
of  the  hydrolysis  of  casein  in  milk  at  36  degrees  (in  the  presence 
of  excess  of  toluol)  is  0.000546,  common  logarithms  being  em- 
ployed and  the  time  expressed  in  hours  (using  the  monomolecular 
formula,  Cf.  previous  chapter  equation  (i)).  For  a  2.8  per  cent 
solution  of  casein  in  NaOH,  carefully  neutralized  to  litmus  and 
therefore  containing  H+  and  OH'  ions  in  the  concentrations  in 
which  they  exist  in  pure  water,  the  velocity-constant  at  36  de- 
grees, similarly  estimated,  proved  to  be  0.000518.  In  the  first 
experiment  the  extent  of  hydrolysis  was  estimated  by  deter- 
mining the  residue  of  undigested  casein  after  32  days,  in  the 
second  experiment  the  undigested  residue  was  determined  after 
20  days.  Now  in  the  second  experiment,  at  all  events,  no  fer- 
ments were  present  and  in  both  experiments  the  solutions  were 
almost  exactly  neutral.  We  must  therefore  regard  the  observed 
hydrolysis  as  being  not  due  to  catalysors  but  to  the  action  of  the 
solvent  water  itself  or  of  its  ions.*  Expressed  in  numerical 
terms,  the  above-cited  results  mean  that  in  absolutely  neutral 
solution,  in  the  absence  of  any  proteolytic  enzymes,  one-half  of 
a  caseinate  of  sodium  or  calcium  is  hydrolysed  in  about  24  days. 

The  hydrolysis  of  neutral  caseinates  by  trypsin  or  pepsin 
affords,  therefore,  an  unusually  favorable  example,  among  pro- 
tein reactions,  of  the  action  of  an  enzyme  in  accelerating  an 
already  progressing  reaction. 

*  Tt  might  be  inferred  from  these  results  that  normal  milk  contains  no 
proteolytic  enzymes.  This  inference  would  not  be  altogether  a  safe  one,  how- 
ever, since  the  milk  which  was  employed  was  obtained  in  the  open  market  and 
there  is  reason  to  suspect  that  it  had  been  manipulated  in  a  manner  which,  it 
is  possible,  may  have  destroyed  pre-existing  enzymes.  No  difficulty  was  en- 
countered in  keeping  this  milk  sterile,  throughout  the  course  of  the  experi- 
ments, by  the  simple  addition  of  toluol.  When  milk  obtained  from  the 
University  Experimental  Farm  was  employed,  however,  which  had  not  been 
pasteurized,  it  was  found  impossible  to  keep  the  milk  sterile,  for  the  length 
of  time  required,  without  employing,  as  sterilizing  agents,  substances  which 
might  conceivably  destroy  or  injure  proteolytic  enzymes. 


PROTEOLYTIC  ENZYMES  AS  CATALYSORS      391 

The  autohydrolysis  of-  the  casemates  in  neutral  and  faintly 
alkaline  solutions  has  been  more  extensively  studied  by  E.  H. 
Walters  (147)  who  has  shown  that  the  neutral  caseinates  of 
lithium,  sodium  and  potassium  in  sterile  solution  undergo  com- 
paratively rapid  autohydrolysis,  approximately  five  per  cent  of 
the  protein  being  hydrolysed  in  ninety-six  hours  at  37.5°  C. 
The  "basic"  caseinates  (neutral  to  phenolphthalein)  of  the  same 
bases  undergo  autohydrolysis  at  a  slightly  higher  velocity.  The 
velocity  of  the  autohydrolysis  of  the  "basic"  casemates  of  cal- 
cium and  barium  is  about  three  times  as  great  as  that  of  the 
autohydrolysis  of  the  caseinates  of  lithium,  sodium  or  potassium, 
indicating  very  clearly  that  some  factor  other  than  the  H+  or 
OH'  ions  plays  a  part  in  determining  the  velocity  of  the 
autohydrolysis. 

The  proteolytic  enzymes  have  long  been  regarded,  by  the 
majority  of  investigators  *  as  true  catalysors;  but  the  evidence 
which  has  been  brought  forward  in  support  of  or  in  opposition 
to  this  view  has  varied  very  much  in  character  from  time  to 
time.  With  the  rapid  gain  in  exact  knowledge  in  the  field  of 
physical  chemistry  which  characterized  the  scientific  advance 
in  the  latter  part  of  the  last  century,  the  characteristic  features 
of  many  catalytic  processes  became  very  thoroughly  known  and 
certain  catalytic  reactions  came  to  be  regarded  as  "typical" 
for  no  better  reason  than  that  they  were  the  best  studied  and 
therefore  the  best  known.  Now  these  so-called  "typical"  in- 
stances of  catalysis  had  in  the  first  place  attracted  the  attention 
of  investigators  simply  because  of  the  marked  peculiarities  which 
they  exhibited  and  which  appeared  to  differentiate  them  sharply 
from  other  chemical  reactions,  and  thus,  by  a  not  unnatural 
process  of  circular  reasoning  these  peculiarities  came  to  be  re- 
garded as  "typical"  and  diagnostic  of  "true"  catalysis.  Our 
conceptions  and  definitions  of  "catalysis"  liave  therefore  altered 
and  enlarged  as  the  number  of  "typical"  instances  has  grown, 
and,  correspondingly,  features  in  the  mode  of  action  of  ferments 
have  been  brought  forward,  at  one  time  in  support  of  the  thesis 
that  the  ferments  are  not  true  catalysors,  and  at  another  time 
in  support  of  the  exact  converse  of  this  view.  Unquestionably 
attempts  have  been  made  by  many  authors  to  shape  the  phe- 
nomena of  fermentation  into  accord  with  prematurely  rigid 
*  Starting  with  Berzelius  and  Liebig.  Cf.  Jacob  Berzelius  (8). 


392  CHEMICAL  DYNAMICS 

conceptions  of  catalysis  based  upon  insufficiently  extended  inves- 
tigation. 

A  phenomenon  which  at  a  very  early  date  greatly  impressed 
the  investigators  in  this  field  was  the  extraordinarily  small  quan- 
tity of  a  catalytic  agent  which,  in  " typical"  instances  of  catalysis, 
was  found  sufficient  to  bring  about  the  chemical  alteration  of 
enormous  quantities  of  material.  Thus  T%  of  a  milligram  of 
colloidal  platinum  will  bring  about  the  combination  of  the  hydro- 
gen and  oxygen  in  no  less  than  10  litres  of  gas,  without  the  least 
reduction  in  its  efficiency  as  a  catalysor  (25);  0.000001  grams  of 
potassium  permanganate  in  10  cc.  of  solution  notably  accelerates 
the  reduction  of  mercuric  chloride  by  oxalic  acid  (64);  the  rate 
of  oxidation  of  an  aqueous  solution  of  sodium  sulphite  is  per- 
ceptibly accelerated  by  the  presence  of  0.0000000000001  N  CuSO4, 
or  even  by  merely  dipping  a  strip  of  clean  metallic  copper  in 
the  water  for  less  than  a  minute  (136).  Were  any  appreciable 
proportion  of  the  catalyst  used  up  during  the  process  of  catalysis 
it  is  obvious  that  quantities  so  minute  as  these  would  be  incapable 
of  bringing  about  the  conversion  of  such  enormous  quantities  of 
material,  and,  in  fact,  we  find  in  many  cases,  even  when  relatively 
large  quantities  of  catalysor  are  employed,  the  catalysor  is  not 
appreciably  used  up  during  the  progress  of  the  reaction;  thus 
a  solution  of  cane  sugar  contains  the  same  amount  of  acid  after 
hydrolysis  as  it  did  before  (19)  (67).  But  if  the  catalysor  is 
unaltered  at  the  end  of  the  reaction  which  it  accelerates  then  it 
must  have  accomplished  this  acceleration  without  adding  any  energy 
to  or  subtracting  any  energy  from  the  reacting  system,  in  other 
words  it  cannot  in  any  way  have  affected  the  final  equilibrium 
of  the  system,  but  only  hastened  the  attainment  of  that  equi- 
librium, since  any  shift  in  equilibrium  must  in  general  be  ac- 
companied either  by  an  expenditure  or  an  absorption  of  energy. 
This  theoretical  deduction  has  been  confirmed  in  a  great  variety 
of  instances  in  the  field  of  non-fermentative  catalysis*;  in  the 
field  of  fermentations  data  bearing  upon  this  deduction  are  either 
lacking  or  fail  to  establish  its  validity  (27)  (14). 

If,  however,  the  station  of  equilibrium  in  any  reaction  is  un- 
affected by  the  presence  of  a  given  catalysor,  then  that  catalysor 

*  Cf.  especially  Turbuba  (137)  (138),  who  has  shown  that  the  equilibrium 
between  aldehyde  and  paraldehyde  is  the  same  whether  sulphur  dioxide,  zinc 
sulphate,  hydrochloric  acid,  oxalic  acid  or  phosphoric  acid  is  used  as  catalysor. 


PROTEOLYTIC  ENZYMES  AS  CATALYSORS      393 

must  accelerate,  and  accelerate  equally,  the  attainment  of  that 
equilibrium  from  either  side;  it  must  in  other  words  accelerate 
the  forward  and  the  reverse  reactions  equally.  This  is  readily 
seen  when  we  reflect  that  equilibrium  in  the  reacting  system  is 
attained  when  the  velocity  of  the  forward  is  equal  to  that  of  the 
reverse  reaction.  If  the  velocity  of  the  forward  reaction  is 
increased  by  any  agency,  therefore,  and  the  station  of  equi- 
librium is  unaffected,  the  velocity  of  the  reverse  reaction  must 
also  have  been  increased  and  in  the  same  proportion.  The 
correctness  of  this  theoretical  deduction  has  also  been  established 
in  a  number  of  "typical"  instances  of  non-fermentative  catalyses, 
but  although  it  has  been  shown  that  yeast  (58)  (59),  kephirlactase 
(28),  diastase  (22),  emulsin  (24),  lipase  (7)  (65)  (55)  (9)  (130) 
(132)  (98)  (99),  trypsin  (135)  and  pepsin  (108)  (109)  (111)  (112) 
(35)  will  not  only  accelerate  the  hydrolysis  of  maltose,  lactose, 
glycogen,  amygdalin,  fats,  protamin  and  paranuclein,  respec- 
tively, but  also  the  synthesis  of  these  substances  from  the  prod- 
ucts of  their  hydrolysis,  yet  it  has  in  no  instance  been  shown 
that  the  velocity  of  the  reverse  reaction  is  accelerated  in  the 
same  proportion  as  that  of  the  forward  reaction;  indeed  such 
evidence  as  exists  tends  to  show  that  this  is  not  the  case  (14) 

CUD. 

From  the  study  of  a  limited  number  of  instances  of  non- 
fermentative  catalysors,  however,  it  appeared,  as  I  have  said, 
that  the  catalysors  are  substances  which  remain  unchanged  at 
the  end  of  the  reaction  which  they  accelerate  and  which  do  not 
shift  the  station  of  equilibrium  and  therefore  cannot  initiate, 
but  only  accelerate  reactions  (89)  (90)  (91)  (92)  (93). 

Recent  investigations,  particularly  those  of  Stieglitz  (127)  (128) 
and  Euler  (27),  have  shown  that  the  above  definition  of  catalysis 
is  too  rigid  and  that  the  cases  which  it  adequately  covers  are 
merely  extreme  instances  of  a  much  more  general  phenomenon. 

It  has  long  been  known  that  in  many  instances  of  unquestioned 
catalysis  the  acceleration  of  the  reaction  in  question  is  accom- 
plished, or  at  least  accompanied  by,  the  formation  of  inter- 
mediate compounds  between  the  substrate  and  the  catalysor. 
The  classic  illustration  of  a  catalysed  reaction  of  this  type  is 
that  afforded  by  the  "continuous  etherification  process"  for  the 
production  of  ether  from  alcohol.  In  this  process  sulphuric 
acid  is  employed  as  catalysor  and,  as  is  well  known,  the  sulphuric 


394  CHEMICAL  DYNAMICS 

acid  first  combines  with  the  alcohol,  forming  a  substance  "which 
can  be  isolated,  namely  ethyl-sulphuric  acid  —  and  this  compound 
reacts  with  another  molecule  of  alcohol,  forming  ether  and  re- 
generating the  sulphuric  acid,  in  accordance  with  the  equations 

HOX  C2H50. 

C2H5OH  +  S02  =  H.OH  +  S02 


HOV 
S02  +  C2H5OH  =  S02  +  C2H5  -  O  -  C2H5 


and,  at  the  end  of  the  reaction,  the  sulphuric  acid  molecule  which 
is  set  free  is  unaltered  and  can  carry  another  molecule  of  alcohol 
through  the  same  series  of  transformations.  Hence,  as  in  the 
"typical"  cases  of  catalysis  cited  above,  the  only  things  which 
prevent  the  reaction  from  proceeding  indefinitely,  with  the  con- 
version of  an  indefinite  quantity  of  alcohol  through  the  agency 
of  a  limited  quantity  of  sulphuric  acid,  are  the  failure  of  the 
supply  of  alcohol,  or  the  continued  accumulation  of  the  products; 
thus,  if  the  water  which  is  formed  in  the  above  reaction  be  allowed 
to  remain  in  the  system  the  sulphuric  acid  becomes,  finally,  so 
highly  diluted  that  the  velocity  of  the  transformation  sinks  to 
a  negligible  magnitude,  i.e.,  for  practical  purposes,  the  reaction 
ceases. 

Another  example  of  the  same  kind  is  the  use  of  nitric  oxide 
as  a  "carrier"  of  oxygen  in  the  manufacture  of  sulphuric  acid. 
The  first  reaction  is  the  combination  of  NO  with  O  to  form 

nitrogen  peroxide: 

2  NO  +  O2  =  2  NO2. 

This  is  brought  into  contact  with  SO2  and  steam,  sulphuric  acid 
is  formed  and  nitric  oxide  regenerated  : 

S02  +  H20  +  NO2  =  H2S04  +  NO 

and  the  NO  is  now  free  again  to  "carry"  oxygen  into  the  sul- 
phurous, converting  it  into  sulphuric  acid. 

In  a  large  number  of  instances  in  which  the  intermediate 
reactions  have  not  been  defined  and  their  occurrence  established, 
it  has  nevertheless  been  shown  that  the  catalysor  forms  com- 
pounds with  the  substrate  or  with  modifications  of  the  substrate, 


PROTEOLYTIC  ENZYMES  AS  CATALYSORS      395 

of  such  a  character  as  to  strongly  suggest  that  they  play  an  im- 
portant part  in  determining  the  course  of  the  reaction.  Thus 
in  the  presence  of  aluminium  chloride,  sulphuryl  chloride  and 
sulphur  chloride  react  thus : 

S02C12  +  S2C12  =  S02  +  2  SC12 

and  it  appears  that  the  aluminium  chloride  aids  the  reaction 
through  the  formation  of  double  compounds  with  the  various 
molecular  species  which  participate  in  the  reaction,  for  many 
such  compounds  are  known,  for  example  A12C12  •  2  S02,  AlClsSCU, 
and  so  forth  (20). 

A  very  decisive  series  of  instances  of  catalysis  through  the 
formation  of]  intermediate  compounds  has  been  established  by 
the  painstaking  and  extensive  researches  of  Stieglitz  (1)  (127) 
(128).  In  the  presence  of  acids  the  iminoesters  (in  aqueous 
solution)  are  decomposed  into  ammonia  and  the  corresponding, 
sparingly  ionized  organic  acid.  It  has  been  shown  by  Stieglitz, 
with  the  utmost  quantitative  precision,  that  the  catalytic  action 
of  the  mineral  acid,  in  this  reaction,  is  accomplished  through 
the  formation  of  salts  of  the  catalysing  acid  with  the  ester,  since 
the  molecular  species  which  actually  undergoes  hydrolysis  in 
this  system  is  the  ester-ion  and  the  salts  of  the  ester  are  much  more 
completely  electrolytically  dissociated  than  the  ester  itself,  so  that 
the  active  mass  of  the  substrate  is  increased  by  the  presence  of  the 
catalysor.  Similarly,  in  the  catalytic  decomposition  of  methyl 
acetate  by  acids  an  oxonium  salt  of  the  ester  with  the  acid  is 
formed  and  it  is  the  positive  ion  of  this  salt  which  undergoes 
hydrolysis.  Now  this  latter  is  a  "typical"  instance  of  catalysis. 
The  catalytic  agent  is  not  used  up  during  the  reaction,  it  can  be 
recovered  from  the  system  unaltered  when  the  reaction  is  com- 
plete, and  the  equilibrium  of  the  reaction  is  not  measurably 
disturbed  by  the  presence  of  the  catalysor.  Nevertheless,  in  a 
comprehensive  mathematical  analysis  of  the  conditions  which 
must  actually  obtain  in  such  a  system,  Stieglitz  has  shown  that 
the  point  of  equilibrium  must  be  shifted  by  the  catalysor,  albeit 
to  an  immeasurably  slight  degree,  and  that  in  the  specific  instance 
under  consideration  the  equilibrium  is  only  inappreciably  shifted 
simply  because  the  quantity  of  the  compound  which  is  formed 
at  any  moment,  between  the  catalysor  and  the  substrate,  is 
evanescently  small,  since  these  salts  are  subject  to  very  extensive 


396  CHEMICAL  DYNAMICS 

hydrolytic  dissociation.  When  the  salt  formation  is  at  any 
instant  extensive  then,  as  Stieglitz  points  out,  although  in  every 
respect  the  mechanism  of  the  acceleration  of  the  hydrolysis  by 
the  catalysing  acid  is  the  same,  yet  a  definite  shift  in  equilibrium 
results  from  the  presence  of  the  catalysor  and,  concurrently,  the 
catalysor  is  in  some  measure  used  up  during  the  progress  of  the 
reaction  and  some  energy  has  to  be  expended  in  order  to  recover 
it,  unaltered,  from  the  system  in  its  final  condition  of  equilibrium. 
I  quote  from  Stieglitz'  article :  * 

"In  accordance  with  the  results,  our  views  concerning  catalytic 
action  must  be  modified  in  regard  to  all  three  of  the  commonly 
assumed  fundamental  characteristics  of  catalytic  action,  (1)  that 
the  acceleration  must  be  proportionate  to  the  concentration  of 
the  catalytic  agent  present;  (2)  that  the  agent  must  not  appear 
to  combine  with  any  of  the  substances  undergoing  change;  and 
(3)  that  the  ultimate  condition  of  equilibrium  must  not  be 
measurably  modified  by  the  presence  of  a  catalysor.  These 
characteristics  are  practically  true  only  for  limiting  cases  where 
the  amount  of  salt-formation  is  so  small  as  to  be  beyond  the 
scope  of  our  measurements.  None  of  them  is  absolutely  true 
under  any  conditions.  When  the  amount  of  salt-formation 
becomes  measurable,  as  for  iminoesters,  they  need  not  hold  even 
approximately,  and  still  the  fundamental  mechanism  and  mode 
of  the  catalysis  is  the  same  in  these  cases  as  in  the  others.  The 
one  vital  fact,  then,  of  an  acceleration  due  to  an  increase  in  the 
active  mass  or  concentration  of  a  reacting  component  in  a  cata- 
lytic action  is  the  only  fundamental  fact  common  to  all  catalytic 
actions. " 

With  this  enlarged  conception  of  catalysis  in  mind  we  need 
no  longer  have  scruples  in  regarding  the  fermentative  reactions 
as  instances  of  true  catalysis.  They  exhibit  the  essential  phe- 
nomenon of  acceleration  through  the  presence  of  a  chemical 
agency  which  is  added  to  the  system,  in  other  words  the  ferment. 
Our  task  is  therefore,  not  to  compare  the  fermentative  reactions 
with  a  special  and  arbitrarily  chosen  class  of  catalytic  reactions 
but,  as  in  all  instances  of  catalysis,  to  attempt  to  unravel  the 
chemical  mechanism  of  the  observed  acceleration. 

In  a  system  so  complex  as  that  afforded  by  a  protein  mixed 
with  an  enzyme  in  aqueous  solution  several  possibilities  present 
*  Julius  Stieglitz  (127).  Cf.  also  Hans  Euler  (26). 


PROTEOLYTIC  ENZYMES  AS  CATALYSORS      397 

themselves,  which  are  too  frequently  overlooked,  and  which 
doubtless  play  a  part  in  determining  the  complexity  of  the 
phenomena  which  are  experimentally  observed. 

In  the  first  place,  we  have  seen  that  certain  of  the  proteins, 
or  protein  salts,  undergo  hydrolysis  at  a  measureable  rate  in 
the  absence  of  proteolytic  enzymes.  Now,  from  the  principle  of 
the  mutual  independence  of  different  reactions  (21)  (53)  (82) 
it  follows  that  when  the  proteins  are  acted  upon  by  proteolytic 
enzymes  both  the  catalysed  and  the  uncatalysed  reactions  must 
be  proceeding  side  by  side,  albeit  the  latter,  possibly,  at  a  re- 
duced velocity.*  Hence  if  the  catalysed  reaction  is  not  over- 
whelmingly more  rapid  than  the  uncatalysed  reaction  the  prog- 
ress of  the  latter  must  disturb  the  time-  and  mass-relations  of  the 
former.  The  velocity-constant  which  is  actually  measured  will 
be  the  sum  of  the  constants  for  the  catalysed  and  the  uncatalysed 
reactions;  only  the  former  constant  will  bear  a  specific  relation 
to  the  mass  of  the  catalysor;  the  sum  of  the  constants,  which 
would  be  the  quantity  actually  measured,  would  not,  therefore 
bear  this  relation  to  the  mass  of  the  catalysor,  but  exhibit  de- 
partures from  it  of  greater  or  less  magnitude  according  to  the 
magnitude  of  the  constant  for  the  uncatalysed  reaction. 

In  the  second  place,  we  have  seen,  in  discussing  the  hydrolysis 
of  the  polypeptids,  that  a  tripeptid  may  undergo  hydrolysis  by 
splitting  at  either  linkage,  and  that  the  hydrolysis  due  to  the 
splitting  of  the  one  linkage  may  be  accelerated  by  one  proteo- 
lytic enzyme,  and  that  due  to  the  splitting  of  the  other  linkage 
by  another  enzyme.  In  so  complex  a  peptid  as  a  protein  many 
linkages  vulnerable  to  the  enzyme  under  consideration  must 
exist  and  it  is  entirely  within  the  bounds  of  probability  that  any 
given  enzyme  shares  its  attack  between  two  or  more  vulnerable 
linkages  and  that,  consequently,  what  we  directly  observe  is  not 
the  progress  of  one  definite  reaction,  or  even  a  catenary  series 
of  reactions,  but  a  number  of  parallel  chains  of  successive  hydrol- 
yses.  On  the  other  hand,  we  know  that  the  vulnerability  of  the 
various  linkages  in  the  protein  molecule  to  various  proteoclastic 
enzymes,  differs  so  that  one  group  of  linkages  will  be  preferen- 
tially attacked  by  one  enzyme,  and  another  group  by  yet  another 
enzyme,  while  no  enzyme  or  group  of  enzymes  will  open  all  of  the 

*  Owing  to  reduction  of  the  active  mass  of  the  free  protein  through  the 
formation  of  a  protein-ferment  compound. 


398  CHEMICAL  DYNAMICS 

—  COHN  —  linkages  which  are  susceptible  to  hydrolysis  by  acids 
(29)  (114)  (56)  (2).     Hence  if  the  enzyme-preparation  which  we 
employ  is  impure,  i.e.,  contains  an  admixture  of  proteolytic  en- 
zymes, then  the  number  of  possible  parallel  reactions  and  number 
of  simultaneous  points  of  attack  in  the  protein  molecule  must  be 
greatly  enhanced.     It  is  probably  to  this  fact  that  we  must  in 
part  attribute  the  much  greater  activity  of  organ-extracts  and  of 
organs  and  tissues  in  situ  than  of  isolated  ferments  in  bringing 
about  the  hydrolysis  of  proteins  (133). 

In  the  third  place  if  the  proteolytic  enzymes  are  proteins  or 
peptids,  as  in  many  cases  seems  highly  probable,  they  must  be 
subject  to  hydration  and  dehydration,  just  as  the  proteins  are, 
and  in  any  case  we  know  that  they  are  thermolabile  and  subject 
in  aqueous  solutions  to  loss  of  activity,  which  is  usually  attributed 
to  hydrolysis,  and  to  modifications  leading  to  precipitation  fol- 
lowing dehydration  by  concentrated  inorganic  salts.  The  pos- 
sibility is  thus  indicated  that  the  proteolytic  ferments  may  exist 
in  two  or  more  modifications,  nor  can  we  assume  that  only  one 
of  these  modifications  is  capable  of  reacting  with  the  substrate, 
or  its  products,  and  influencing  the  velocity  with  which  they 
undergo  modification.  The  activity  of  the  ferment  in  acceler- 
ating either  hydrolysis  or  synthesis  of  protein  may  therefore 
vary  with  the  conditions  under  which  it  acts  and  among  these 
conditions  must  be  reckoned  both  the  relative  and  the  absolute  con- 
centrations of  the  substrate  and  products  of  the  reaction,  since  they 
must  modify,  through  combination  with  them,  the  equilibrium 
between  any  different  forms  of  the  enzyme  which  may  exist 
in  the  system.  In  quite  general  terms,  however,  and  without 
assuming  the  existence  of  more  than  one  form  of  the  ferment, 
it  may  be  stated  that  if  the  ferment  enters,  at  any  stage  of  the 
reaction  of  protein-hydrolysis,  into  combination  with  the  sub- 
strate to  any  appreciable  extent  (and  we  shall  see  that  in  many 
instances  it  does),  since  the  final  equilibrium  of  the  reaction  must 
therefore  be  affected  by  the  ferment,  the  ferment  must  to  some 
extent  be  used  up  in  the  hydrolysis  and  the  activity  of  the  ferment 
must  vary  as  the  reaction  proceeds. 

2.  The  Evidence  for-  the  Existence  of  Intermediate  Com- 
pounds between  the  Proteolytic  Ferments  and  their  Substrates. 

—  The  existence  of  compounds  between  proteins  and  proteolytic 
enzymes  has  been  established  in  a  number  of  cases  by  a  variety 


INTERMEDIATE  COMPOUNDS  399 

of  observers.  Thus  Vernon  (145)  has  shown  that  serum  albumin, 
paraglobulin  and  particularly  egg-albumin,  when  added  to  a 
tryptic  digest,  markedly  delay  the  hydrolysis  of  another  protein 
by  the  trypsin.  Obviously,  if  the  enzyme  did  not  enter  into 
combination  with  its  substrates,  the  rate  at  which  it  attacks 
one  substrate  should  not  be  influenced  by  the  presence  of  another. 
Hedin  (46)  has  shown  that  the  power  of  egg-albumin  to  delay  the 
hydrolysis  of  another  protein  by  trypsin  is  much  greater  if  the 
egg-albumin  and  the  trypsin  are  mixed  together  before  the  trypsin 
is  added  to  the  digest  than  after.  Evidently  the  egg-albumin- 
trypsin  compound  is  but  slowly  reversible;  when  none  of  the 
trypsin  is  bound  by  another  substrate  the  active  mass  of  free 
trypsin  is  greater  and  the  proportion  bound  by  albumin  is  there- 
fore greater.  Once  the  trypsin  is  bound  by  albumin  it  is  only  very 
slowly  abstracted  from  the  combination  by  another  substrate.* 

Dauwe  (23)  has  shown  that  trypsin  can  be  extracted  from  its 
solution  by  coagulated  egg-white  or  by  fibrin,  and  the  ferment 
can  be  regained  from  the  compound  by  prolonged  washing  with 
water.  From  what  has  been  said  in  Chap.  V,  section  3,  it  will  be 
clearly  realized  that  this  latter  fact  does  not  in  the  least  militate 
against  the  view  that  the  combination  between  the  ferment  and 
the  substrate  is  chemical  in  character. 

A  very  striking  instance  of  the  way  in  which  the  formation 
of  compounds  of  this  type  may  interfere  with  the  time-  and  mass- 
relations  in  the  main  hydrolysis  under  observation  has  been 
discovered  by  Hedin  (49).  When  trypsin  acts  upon  casein,  the 
time  required  for  the  attainment  of  a  given  degree  of  hydrolysis 
(=  0  is  inversely  proportional  to  the  concentration  of  ferment 
(=  p).  In  other  words  pt  =  constant.  But,  if  egg-albumin  be 
present,  a  proportion  of  the  trypsin  is  bound  by  the  albumin 
and  this  proportion  is  greater  the  more  dilute  the  ferment,  so  that 
the  quantity  of  ferment  which  is  free  to  act  upon  the  trypsin 
is  no  longer  proportionate  to  its  concentration  and  the  law  no 
longer  holds  good.  From  this  it  is  clear  that  if  we  wish  to  obtain 
readily  interpretable  time-  and  mass-relations  in  a  protein  digest 
we  must  employ  only  pure  proteins.  We  have  seen,  also,  that  we 

*  Hedin  has  also  shown  that  a  greater  proportion  of  trypsin  is  bound  at 
higher  than  at  lower  temperatures.  But  if  excess  of  trypsin  is  attached  to  the 
albumin  by  temporarily  raising  the  temperature,  on  lowering  the  temperature 
the  trypsin  is  not  given  up  again. 


400  CHEMICAL  DYNAMICS 

must  employ  only  a  single  enzyme.  Anyone  who  possesses  an 
extensive  knowledge  of  the  literature  on  protein  hydrolysis  by 
enzymes  will  readily  admit  that  these  conditions  have  very  rarely 
been  realized. 

3.  The  Kinetics  of  Protein  Hydrolysis  by  Enzymes.  —  In 
several  instances  it  has  been  found  that  very  small  quantities 
indeed  of  proteolytic  enzyme  will  suffice  to  convert  large  quan- 
tities of  protein;  thus  one  part  of  rennet  will  curdle  from  400,000 
to  800,000  parts  of  milk  (42)  and  a  pepsin  powder  has  been  pre- 
pared which  in  seven  hours  dissolved  500,000  times  its  weight 
of  fibrin.  From  these  facts  it  has  been  urged  by  many  observers 
that  the  fermentative  splitting  of  proteins  is  an  instance  of 
"typical"  catalysis,  in  the  limited  sense  of  the  term  described 
in  section  1.  The  justification  for  this  view  is  totally  inadequate, 
for  it  has  not  been  shown  that  at  the  end  of  extensive  hydrolysis 
the  enzyme  can  be  recovered  from  the  digest,  wholly  unaltered 
in  efficiency,  without  the  expenditure  or  absorption  of  any 
energy.  In  fact,  in  many  cases  it  is  known  that  the  activity  of 
the  enzyme  is  impaired  during  the  hydrolysis.  Since  this  im- 
pairment of  activity  is  even  more  pronounced  when  proteins 
are  not  present,  it  is  usually  conceded  that  autohydrolysis  of 
the  enzyme  itself  suffices  to  account  for  the  whole  of  this  effect; 
this  is  clearly  not  necessarily  the  case;  while  autohydrolysis  of 
the  enzyme  renders  it  very  difficult  to  ascertain  whether  the 
enzyme  is  actually  exhausted  by  the  hydrolysis  or  not,  yet  it  does 
not  exclude  the  possibility  that  such  exhaustion  occurs.  Now 
it  must  be  constantly  borne  in  mind  that  the  proteins  are  very 
nearly  thermoneutral  substances;  their  hydrolysis  (Cf.  section  1) 
is  accompanied  by  the  disengagement  of  heat,  it  is  true,  but  this 
disengagement  of  heat  is  very  minute.  Hence  the  energy  change 
which  is  involved  in  a  shift  of  the  equilibrium  between  protein 
and  the  products  of  its  hydrolysis  must  be  very  minute  and  a 
shift  in  the  equilibrium  of  a  correspondingly  minute  quantity 
of  a  less  thermoneutral  substance  might  very  well  suffice  to 
provide  or  absorb  the  requisite  energy  to  bring  about  a  very 
decided  shift  in  the  equilibrium  between  protein  and  its  prod- 
ucts. We  cannot  assume,  therefore,  from  the  smallness  of  the 
amount  of  enzyme  which  will  transform  large  quantities  of  pro- 
tein, that  the  enzyme  does  not  exert  any  influence  upon  the 
equilibrium  which  the  protein  finally  attains  with  its  products. 


KINETICS  OF  PROTEIN  HYDROLYSIS  401 

If  we  assume,  however,  (i)  that  the  ferment  does  not  enter 
appreciably  into  combination  with  the  substrate  or  products 
and  hence  does  not  shift  the  equilibrium  between  the  protein 
and  its  products,  (ii)  that  the  products  of  the  reaction  do  not 
appreciably  depress  the  velocity  of  hydrolysis,  and  (iii)  that 
the  reaction  of  the  digest  either  does  not  affect  the  velocity  of 
hydrolysis  or  is  unaltered  by  its  progress,  then  for  a  certain  period 
of  the  hydrolysis,  we  may  regard  the  transformation  as  concerning, 
on  the  whole,  only  one  molecular  species,  and  the  equation 

log^  =  fc  (i) 

should  hold  good. 

It  will  be  well,  before  proceeding  further,  to  clearly  understand 
what  is  implied  in  the  statement  that  this  equation  will  hold 
good  for  a  certain  period  of  the  hydrolysis.  When  the  first 
—  COH.N—  bond  in  the  protein  molecule  splits  (considering, 
for  the  present,  only  one  of  the  possible  parallel  reactions,  since, 
in  a  system  of  parallel  reactions  of  the  same  order,  the  reaction- 
constants  of  the  different  reactions  simply  add  themselves  to- 
gether to  produce  the  gross  resultant)  a  product  p,  is  the  result. 
This  product,  however,  now  becomes  the  substrate  for  a  second 
reaction  involving  the  splitting  of  another  —COH.N—  bond 
and  the  production  of  a  second  substance  pz.  Two  possibilities 
now  exist.  The  transformation  pi  — *•  pz  may  be  specifically  more 
rapid  than  the  transformation  p  — >  p±  or  it  may  be  specifically 
less  rapid.  In  either  case  it  will  initially  be  in  an  absolute  sense 
less  rapid  than  the  primary  reaction  since  its  substrate-concen- 
tration is  initially  zero  and,  consequently,  the  absolute  velocity  of 
the  second  reaction  will  rise  until  it  attains  a  maximum  value,  and 
the  combined  reactions  will  then  proceed  with  the  velocity  and 
time-relations  of  the  specifically  slowest  reaction.  If  this  should 
chance  to  be  the  primary  reaction  then  equation  (i)  will  hold 
throughout;  if  it  should  chance  to  be  the  secondary  reaction 
then  equation  (i)  will  hold  after  the  secondary  reaction  has  attained 
its  maximum  velocity  but  not  before. 

The  meaning  and  derivation  of  equation  (i)  will  be  clear  when 
it  is  recollected  that  the  reaction  of  protein  hydrolysis  consists 
in  the  addition  of  a  molecule  of  water  to  a  —COH.N—  bond 
in  the  protein  molecule.  In  dilute  solutions  the  active  mass  of 


402  CHEMICAL  DYNAMICS 

water  will  not  be  appreciably  affected  by  the  inclusion  of  a 
minute  proportion  of  the  water  in  the  products  of  hydrolysis. 
Only  one  molecular  species  is  appreciably  changing  in  concen- 
tration, therefore,  namely  the  protein.  The  number  of  molec- 
ular collisions  per  second  between  the  water  and  the  protein 
molecules  will  therefore  be  proportionate,  at  any  instant,  to  the 
concentration  of  unaltered  protein  at  that  instant.  If  the  initial 
concentration  is  a  and  the  amount  hydrolysed  after  time  t 

is  x,  the  velocity  of  hydrolysis  f-^-J  is  given  by  -r  =  k  (a  —  x), 

in  which  k  is  the  velocity-constant  (specific  velocity,  or  velocity 
per  unit  concentration)  of  the  reaction.  Integrating  this,  and 
recollecting  that  when  t  =  0,  x  =  0  we  obtain  equation  (i) . 

The  simplifying  assumptions  which  we  have  been  compelled 
to  make  in  deriving  this  formula  are  very  numerous  and  very 
many  of  them  devoid  of  either  theoretical  or  experimental  justi- 
fication. Hence  it  is  not  surprising  that  it  has  not  often  been 
found  to  hold  good  by  those  who  have  followed  the  time-relations 
of  protein  hydrolysis. 

Victor  Henri  and  Larguier  des  Bancels  (54)  studied  the  di- 
gestion of  gelatin  and  casein  by  trypsin,  following  the  hydrolysis 
by  observing  the  increase  in  the  conductivity  of  the  digest,  under 
the  assumption  that  each  molecule  of  hydrolysed  protein  con- 
tributes equally  to  the  observed  increase  in  conductivity.  They 
found  that  the  values  of 

7          1  i  a 

k  =  -  log 

t     6a  —  x 

were  tolerably  constant  for  brief  periods  of  digestion  and  for 
varying  values  of  a  (—  initial  substrate-concentration),  in  none 
of  their  experiments,  however,  did  they  approach  the  stage  of 
complete  digestion,  and  Arrhenius  (3)  has  shown  that  their 
results  are  equally  well  expressed  by  the  formula: 

x  =  kiVt  (ii) 

which  is  the  well-known  "rule  of  Schutz"  to  the  effect  that  the 
quantity  of  protein  which  is  digested  by  a  given  quantity  of 
proteolytic  ferment  is  proportional  to  the  square  root  of  the 
period  occupied  in  digestion. 

Bayliss,  also  employing  the  conductivity  method  (5),  studied 
the  hydrolysis  of  casein  by  trypsin.  He  found  that  the  constant, 


KINETICS  OF  PROTEIN  HYDROLYSIS  403 

calculated  from  the  monomolecular  formula,  falls  off  rather 
rapidly  as  digestion  proceeds.  The  decrease  in  the  value  of  the 
constant  is  not  attributable  wholly  to  autodestruction  of  the 
ferment.  Arrhenius  has,  moreover,  shown  that  Bayliss'  results 
are  also  in  satisfactory  accord  with  the  rule  of  Schtitz. 

Taylor  (133),  studying  the  hydrolysis  of  protamin  sulphate 
by  trypsin,  found  a  tolerable  accord  with  the  monomolecular 
law: 

Substrate  0.150.    Vol.  100.    Ferment  0.001.    Temp.  34  degrees, 
time  =  15     30    45     60    75    90     105     150 

k  X  104  68     69     69     76    66    64      62      60    Average  =  67 

Substrate  0.100.    Vol.  100.     Ferment  0.001.     Temp.  34  degrees, 
time  =  15     30    45     60    75     90     105     150 

k  X  104  79     84    80    76    73    70    64        68    Average  =  74 

Substrate  0.0075.     Vol.  100.     Ferment  0.001.     Temp.  34  degrees, 
time  =  15     30    45    60    75    90     105     150 

k  X  104  90     97    92    88    83    88      79      74    Average  =  86 

but  it  will  be  observed  that  the  constant  rises  in  value  as  the 
substrate-concentration  decreases.  Therefore,  as  Taylor  points 
out,  in  a  physical  sense  the  direct  proportionality  between  the 
velocity  of  hydrolysis  and  the  concentration  of  unhydrolysed 
protein  "is  only  spurious,  since  it  holds  but  for  each  particular 
system." 

On  the  other  hand,  Samojlov  (116),  Walter  (148),  Sjoquist  (125), 
Schiitz  and  Huppert  (124),  Gross  (41)  and  Meyer  (79),  working 
with  pepsin  and  employing  a  variety  of  substrates,  have  con- 
curred in  finding  that  for  a  considerable  period  of  digestion  the 
Schiitz  rule  holds  good;  Borissov  (11),  employing  trypsin.  has 
also  confirmed  the  Schiitz  rule  and,  as  we  have  seen,  Arrhenius 
has  shown  that  the  results  of  Bayliss  and  of  Henri  and  des  Bancels 
are  in  accord  with  this  rule.  Weis  (150)  working  with  the 
enzyme  contained  in  malt-extract,  with  wheat-protein  as  sub- 
strate, has  also  confirmed  the  Schiitz  rule,  although  in  very 
dilute  solutions  of  ferment  the  exponent  of  the  time  tended  to 
rise  and  approach  unity,  i.e.,  the  relation  tends  to  become 

x  =  kst.  (iii) 

Arrhenius  (loc.  cit.)  has  shown,  from  the  results  of  Weis, 
that  the  quantity  of  protein  hydrolysed  by  a  given  quantity  of 


404  CHEMICAL  DYNAMICS 

ferment  in  a  given  time  is  directly  proportional  to  the  initial 
concentration  of  the  substrate,  i.e.,  the  true  form  of  equation  (ii)  is 


a  result  which  had  previously  been  established  by  Schiitz  and 
Huppert  (loc.  cit.) 

Arrhenius  (loc.  cit.,  p.  64)  has  pointed  out  that  the  Schiitz 
rule  may  be  derived  from  the  monomolecular  formula  provided 
we  assume  that  the  catalysor  in  the  system  enters  into  combi- 
nation with  and  so  is  inactivated  by  the  products  of  the  reaction. 
This  derivation  is,  however,  only  valid  for  the  early  stages  of 
hydrolysis,  during  which  x  is  small  in  comparison  with  a. 

Regarding  the  dependence  of  the  velocity  of  hydrolysis  upon 
the  ferment-concentration,  Taylor  (loc.  cit.)  finds  that  the  time 
required  to  hydrolyse  a  given  quantity  of  protamin  sulphate  to 
a  given  degree  is  inversely  proportional  to  the  quantity  of  fer- 
ment present.  I  have  found  (107)  that  the  velocity-constant 
of  the  hydrolysis  of  calcium  and  barium  caseinate  in  dilute 
solutions,  also  calculated  from  the  monomolecular  formula,  is 
directly  proportional  to  the  concentration  of  trypsin,  but  at 
higher  substrate  concentrations  (A^/400  Ca(OH)2  neutralized  by 
casein)  the  ratio  k/F  increases  with  increasing  ferment-concen- 
tration. Hedin  (47)  (48)  (49)  has  studied  the  hydrolysis  of 
casein  by  trypsin  very  carefully  from  this  standpoint  and  finds 
that  the  relation 

Ft  =  constant,  (iv) 

where  F  is  the  mass  of  ferment  and  t  the  time  required  to  attain 
a  certain  degree  of  hydrolysis,  holds  good  over  a  wide  range  of 
ferment-concentrations. 

Employing  the  method  of  observing  the  rate  of  solution  of 
coagulated  protein  enclosed  in  capillary  tubes  Schiitz  found 
that  the  rate  of  digestion  is  proportional  to  the  square  root 
of  the  ferment  (pepsin)  -concentration.  Taylor  (loc.  cit.)  and 
Arrhenius  (loc.  cit.)  have,  however,  pointed  out  that  if  this 
method  of  measurement  be  employed,  processes  of  diffusion  and 
solution  are  liable  to  be  confused  with  the  process  of  hydrolysis. 
From  the  results  of  Hedin,  however,  which  are  cited  above, 
since  Ft  =  constant  and  the  rule  of  Schiitz  (x  =  k  V7)  holds 
good  in  a  large  number  of  instances  of  protein  hydrolysis,  it 


KINETICS  OF  PROTEIN  HYDROLYSIS  405 

would  appear  that  in  these  instances,  at  least,  the  extent  of 
hydrolysis  after  a  given  time  must  be  proportional  to  the  square 
root  of  the  ferment-concentration  (41). 

The  influence  of  the  formation  of  compounds  between  the 
substrate  and  the  enzyme  upon  the  kinetics  of  hydrolysis  is  very 
clearly  revealed  by  the  experiments  of  Bogddndy  (10)  who  has 
shown  that  when  a  large  excess  of  substrate  is  present  the  velocity 
of  protein  hydrolysis  by  pepsin  depends  only  upon  the  mass  of 
the  ferment,  while,  when  a  large  excess  of  ferment  is  present, 
it  depends  only  upon  the  mass  of  the  substrate.  This  is  obviously 
what  we  should  expect  to  be  the  case  were  the  actual  substance 
undergoing  hydrolysis  a  compound  of  the  substrate  and  the 
enzyme. 

The  hydrolysis  of  caseinates  by  trypsin  has  been  exhaustively 
investigated  by  Walters  (146)  who  has  shown  that  the  relation 
between  the  time  of  hydrolysis  and  the  amount  of  sodium  casein- 
ate  hydrolysed  is,  for  all  stages  of  the  reaction,  almost  exactly 
what  would  be  expected  from  the  monomolecular  formula.  More- 
over the  velocity  of  hydrolysis  is  directly  proportional  to  the 
concentration  of  trypsin  and  there  is  a  less  exact  proportionality 
between  the  initial  concentration  of  the  substrate  and  the  velocity 
of  hydrolysis,  the  velocity-constant  decreasing  slightly  as  the 
concentration  of  the  substrate  increases.  The  nature  of  the 
base  combined  with  the  casein  has  little  or  no  influence  upon 
the  process  of  hydrolysis  by  trypsin  (although  it  has  a  decided 
influence  upon  the  velocity  of  autohydrolysis  (147))  from  which 
we  may  infer,  since  caseinates  of  the  alkalies  and  of  the  alkaline 
earths  are  hydrolysed  with  equal  velocity,  that  the  degree  of 
electrolytic  dissociation  of  the  caseinate  has  little  or  no  effect 
upon  the  velocity  of  its  hydrolysis  by  trypsin. 

We  thus  see  that  under  different  conditions  of  enzyme-  and 
substrate-concentration  and  with  different  substrates  very  dif- 
ferent laws  are  found  to  express  the  relationship  between  time 
and  the  extent  of  hydrolysis  of  the  proteins  and  polypeptids. 
None  of  these  relations  holds  good  outside  certain  definite  limits 
of  substrate-  and  enzyme-concentration  and  each  of  them  only 
holds  good  for  a  limited  portion  of  the  hydrolysis.  We  are  in- 
clined to  suspect  that  each  of  these  relations  is  but  part  of  some 
more  general  relation  which,  it  is  possible,  holds  good  for  all 
of  the  cases  cited  above  and  for  all  stages  of  the  hydrolysis. 


406  CHEMICAL  DYNAMICS 

The  task  of  seeking  for  such  a  relation,  when  we  recall  all  of  the 
factors  which  may  very  possibly  enter  into  it,  might  well  seem, 
for  purposes  of  practical  utility,  well-nigh  hopeless,  were  it  not 
for  the  fact  that  the  limiting  cases  of  this  general  law  have 
already  been  ascertained  to  hold  good  over  quite  extensive  and 
practically  attainable  ranges  of  time  and  concentration.  Evi- 
dently the  number  of  the  factors  which  play  an  appreciable  part 
in  determining  the  phenomena  observed  is  not  so  great  as  we 
might  be  inclined  to  anticipate. 

We  have  seen  that  Henri's  law  does  not  suffice;  it  is  not 
sufficiently  general  to  cover  all  of  the  time-  and  mass-relations 
in  the  hydrolysis  of  the  polypeptids  (Cf.  the  previous  chapter, 
section  2) ;  we  are  led  to  suspect  that  some  important  factor  has 
been  omitted  in  the  derivation  of  Henri's  equation. 

In  the  following  chapter  we  shall  see  that  pepsin  is  capable 
of  exerting  synthetic  activity  under  conditions  such  that  its 
hydrolytic  activity  is  absent.  We  have  already  had  occasion 
to  indicate  that  the  proteolytic  ferments  may  exist  in  more  than 
one  modification.  In  the  light  of  these  facts  we  shall  make  the 
following  assumptions  regarding  the  mode  of  action  of  the  pro- 
teolytic ferments  upon  the  proteins. 

Let  us  suppose  that  the  proteolytic  ferments  act  as  " carriers" 
of  water  into  the  protein  molecule,  just  as  nitric  oxide,  in  the 
manufacture  of  sulphuric  acid,  acts  as  a  " carrier"  of  oxygen  into 
sulphurous  acid.  We  must  therefore  assume  that  these  ferments 
can  exist  both  in  a  hydrated  and  in  a  dehydrated  form,  just  as 
nitric  acid  can  exist  in  the  reduced  form  of  nitrous  oxide.  More- 
over we  must  conclude  that  at  the  splitting  of  each  —  COH.N  — 
bond  in  the  protein  molecule  some  such  reactions  as  the  following 

occur : 

HF 
I 

(A)  -COH.N-  +  HFFOH  =  -  COH.N  - 

(hydrated  I 

*— °  FOH 

\  ferment-substrate  compound) 

HF 

(B)  -  COH.N  -  -  -  COOH  +  H2N  -  +  FF 

I  (products  of  hydrolysis)      (dehydrated 

FOH 

(ferment-substrate 
compound) 


KINETICS  OF  PROTEIN   HYDROLYSIS  407 

So  far  we  have,  in  essentials,  made  only  two  assumptions,  the 
one  the  original  assumption  that  the  ferment  acts  as  a  ''carrier" 
of  water,  the  other  that  one  molecule  of  ferment  reacts  with  one 
-COH.N-  bond. 
Combining  the  above  two  equations  we  obtain: 

(C)  -COH.N-  +  HFFOH  =  -COOH  +  H2N-  +  FF 

from  which  it  is  evident  that  the  point  of  equilibrium  in  the 
reaction : 

(D)  FF  +  H20  <=»  HFFOH 

must  be  shifted  in  some  measure  towards  the  left  by  the  presence 
of  the  substrate  and  the  extent  of  this  shift  must  bear  a  constant 
proportion  (a)  to  (a  —  x).  But  this  equilibrium  must  also  be 
shifted  towards  the  right  by  the  presence  of  the  products  of  the 
hydrolysis  of  the  protein,  and  this  shift  must  bear  a  constant 
proportion  (0)  to  x2. 

Let  us  now  analyse  the  physical  meaning  of  Henri's  equation. 
This  equation,  as  we  have  seen  in  Chap.  XV,  may  be  written: 

log  — ^—  +  ax  =  kFt,  (v) 

tt  —  X 

in  which  a  and  k  are  constants  and  F  is  the  total  mass  of  ferment 
present  in  the  system. 
Differentiating  this  equation  we  obtain: 

dx  F  7  /          \  /  -\ 

37  =  1-1 1 \  k(a  —  x),  (vi) 

dt      I  +  a  (a  —  x) 

which  means  that  the  actual  "active  mass"  of  the  proteolytic 
ferment,  that  proportion,  namely,  which  accelerates  the  hydrol- 
ysis by  multiplying  the  velocity-constant,  is  not  F  but 

F 

l  +  a(a-x)' 

In  other  words,  the  process  of  combination  between  the  ferment 
and  the  substrate  and  its  products  which  Henri  depicts,  results 
in  the  inactivation  of  a  certain  constant  proportion  of  the  ferment 
by  each  molecule  of  the  substrate.  The  mechanism  of  this  will 
be  clear  from  equation  (C);  by  the  same  equation  it  will  also 
be  clear  that  a  proportion  of  the  ferment  is  at  the  same  time 
activated  (rendered  available  for  the  acceleration  of  the  hydrolysis). 
The  quantity  of  ferment  thus  activated  must  evidently  bear  a 


408  CHEMICAL  DYNAMICS 

constant  proportion  /3  to  x2.     Introducing  this  factor  into  equa- 
tion (vi)  we  obtain: 

dx  F 


/  -x 
(vn) 


Integrating,  we  obtain: 


(1  -  0a2)  log  —  —  +  (a  +  0a)  x  +  ^x*  =  kFt.         (viii) 

a  —  x  £ 

In  the  derivation  of  Henri's  equation  it  is  assumed  either  that 
the  ferment  is  unaltered  during  the  processes  which  accomplish 
the  introduction  of  the  elements  of  water  into  the  protein  mole- 
cule or  else  that  the  equilibrium  in  equation  (D)  lies  so  far  to  the 
right  that  for  all  practical  purposes  the  ferment  is  present  wholly 
in  the  hydrated,  proteolytically  active  form.  Equation  (viii) 
is  Henri's  equation  generalized  to  the  extent  of  including  any 
species  of  equilibrium  in  equation  (D). 
Equation  (viii)  may  be  written  : 

a       ,    a  -f  /So  |8  kFt  ,.  . 

log  J^  +  nr^*  +  2(i-^)x  •  (T=W) 

from  which  it  is  evident  that  it  contains  three  mutually  inde- 
pendent constants.  This  fact  deprives  it  of  any  great  utility  for 
the  purpose  of  comparing  numerical  data  with  the  theoretical 
deductions,  since  an  equation  containing  three  mutually  inde- 
pendent constants  may  be  made  to  fit  with  tolerable  exactitude 
any  continuous  curve  by  an  appropriate  choice  of  constants. 
In  the  absence  of  very  numerous  and  very  exact  data,  a  numerical 
comparison  between  theory  and  experiment  involving  this 
equation,  therefore,  would  be  devoid  of  utility.  It  is  of  interest 
to  observe,  however,  that  all  the  relationships  between  x,  F  and 
t  found  by  the  various  observers,  whose  investigations  we  have 
cited  above  and  in  the  previous  chapter,  are  especial  cases  of  the 
general  relation  which  is  expressed  in  equation  (ix).  Thus  if 
/3  be  small,  that  is,  if  the  shift  in  the  equilibrium  between  the 
hydrated  and  unhydrated  forms  of  the  enzyme  due  to  unit  mass 
of  the  products  of  the  hydrolysis  is  very  small,  or  if  the  equi- 
librium in  equation  (D)  .  lies  far  to  the  right,  then  we  regain 
Henri's  equation.  If  not  only  0  but  a  is  small,  i.e.,  if  the  part 
played  by  the  protein  and  its  products  in  determining  the  equi- 
librium between  the  different  forms  of  ferment  is  small,  then  we 


KINETICS  OF  PROTEIN  HYDROLYSIS 


409 


regain  the  monomolecular  formula.     If  jSa2  is  large  in  comparison 
with  1  and  a  is  small  then  we  obtain  the  relation 

a       .  x  .    x2        kF  . 


which,  when  x  is  small,  yields  the  relation: 
-  =  kFt  (equation  (iii)). 

It  is  obvious  from  equation  (vii)  that  if  the  velocity  constant 
k  were  to  be  calculated  from  the  monomolecular  formula  through- 
out the  reaction  of  hydrolysis,  k  would  tend  to  fall  off  as  hydrolysis 
proceeded,  i.e.,  as  x  increased,  and  also,  it  will  be  evident,  even 
for  small  values  of  x  the  constant  would  decrease  with  increasing 
initial  substrate-concentration.  This  obviously  accords  with  the 
facts  observed. 

The  way  in  which  the  relationship  expressed  in  equation  (ix) 
may  simulate  the  monomolecular  formula,  the  Schiitz  rule,  etc., 
under  certain  conditions,  is  very  well  shown  by  the  following 
table.  The  values  of  t  corresponding  to  various  values  of  x 
are  calculated  from  formula  (ix)  on  the  assumption  that  a  =  10 
and  that  a  \  aa  Q  j^p 

—    —    — =  .  =:    1_^ 

1  —  /3(  ~ 


2  (1  -  /3a2)       ( 

From  these  values  of  t  and  the  given  values  of  x  are  calculated 
the  constants  corresponding  to  the  law  of  direct  proportionality 
between  the  quantity  digested  and  the  time,  to  the  Schiitz  rule 
(t  =  kx2)  and  to  the  monomolecular  formula: 


X 

t 

H 

*=^ 

""-'rh 

0.1 

0.1144 

1.144 

11.44 

26.0 

0.2 

0.2488 

1.244 

6.22 

28.3 

0.5 

0.7723 

1.545 

3.09 

34.6 

1.0 

2.046 

2.046 

2.05 

44.5 

2.0 

6.097 

3.049 

1.52 

62.9 

3.0 

12.155 

4.051 

1.35 

78.4 

•   4.0 

20.222 

5.055 

1.26 

91.1 

5.0 

30.301 

6.060 

1.21 

100.7 

6.0 

42.398 

7.066 

1.18 

106.5 

7.0 

56.523 

8.075 

1.15 

108.1 

8.0 

.  72.699 

9.087 

1.14 

104.0 

9.0 

89.000 

9.889 

1.21 

89.0 

410  CHEMICAL  DYNAMICS 

It  will  be  seen  that  from  x  =  0.1  to  x  =  0.5  the  law  of  direct 
proportionality  very  nearly  holds  good;  from  x  =  3.0  to  x  =  9.0 
the  Schtitz  rule  very  nearly  holds  good,  while  from  x  =  4.0  to 
x  =  8.0  the  monomolecular  formula  adequately  expresses  the 
relationship  between  x  and  t. 

4.  The  Influence  of  Acid  and  Alkali  upon  the  Rate  of  Protein 
Hydrolysis  by  Enzymes.  —  Of  great  importance  in  determining 
the  rate  of  hydrolysis  of  proteins  by  proteolytic  enzymes  is  the 
reaction  (H+  or  OH'  concentration)  of  the  solution  in  which 
digestion  is  occurring.  The  activity  of  pepsin  is  greatly  en- 
hanced by  a  low  degree  of  acidity,  greater  acidity  hindering  its 
activity  through  destruction  of  the  enzyme  itself  by  the  excess 
of  acid.  The  activity  of  trypsin,  on  the  other  hand,  is  very 
markedly  favored  by  a  very  slight  alkalinity,  excess  of  alkali 
being  even  more  destructive  to  trypsin  than  excess  of  acid  is 
to  pepsin.  An  excess  of  acid  very  rapidly  destroys  the  activity 
of  trypsin,  while  an  alkaline  medium  leads  to  the  somewhat 

N 
less  rapid  destruction  of  pepsin.     According  to  Taylor  -—^  to 


N 

OH'  is  the  optimum  alkalinity  for  the  action  of  trypsin 


1400 

upon  protamin  sulphate  (129). 

The  nature  of  the  part  played  by  the  alkali  and  acid  in  protein 
hydrolysis  by  enzymes  is  by  no  means  clear.  Since  acids  and 
alkalies  (H+  and  OH'  ions)  are  well-known  hydrolysing  agents, 
a  very  natural  assumption  regarding  their  influence  upon  protein 
hydrolysis  by  enzymes  is  to  suppose  that  they  play  the  part  of 
accessory  catalysors,  the  catalytic  action  of  the  acid  or  alkali 
being  added  to  that  of  the  enzyme.  As  regards  the  influence 
of  acids  upon  the  hydrolysis  of  proteins  by  pepsin,  however, 
there  are  many  well-known  facts  which  speak  against  this  view. 

The  influence  of  acids  is  by  no  means  proportional  to  their 
degree  of  dissociation  as  would  be  expected  were  their  influence 
due  to  a  catalytic  action  of  the  hydrogen  ion  (6)  (68).  On  the 
contrary,  hydrochloric  acid  has  an  almost  specific  action  upon 
the  hydrolysis  of  proteins  by  pepsin,  so  that  many  observers 
have  inclined  to  the  belief  that  the  real  ferment  is  in  this  instance 
a  compound  of  pepsin  with  hydrochloric  acid  (121).  Loeb  (74) 
has  suggested  that  the  part  played  by  the  acid,  in  accelerating 
the  hydrolysis  of  proteins  by  pepsin,  is  analogous  to  the  part 


INFLUENCE  OF  ACID  AND  ALKALI  411 

played  by  acids  in  accelerating  the  hydrolysis  of  the  imino- 
esters  by  increasing  the  active  mass  of  the  substrate  through  salt 
formation  (Cf.  Stieglitz'  experiments,  cited  in  section  1).  Loeb 
believes  that  the  portion  of  the  pepsin  which  is  active  in  bring- 
ing about  the  hydrolysis  of  proteins  is  that  portion  of  it  which 
is  ionized,  and  that  acids  increase  the  active  mass  of  the  ferment 
by  forming  ionized  salts  with  it.  Referring  to  the  theory  of 
protein  ionization  which  is  developed  in  earlier  chapters  of  this 
work,  and  the  intimate  relation  which,  as  we  have  seen,  subsists 
between  the  degree  of  ionization  and  the  degree  of  hydration 
of  a  protein  (and  presumably  of  these  enzymes)  this  view  would 
appear  very  probable,  especially  if  we  accept  the  account  of  the 
mechanism  of  the  hydrolysis  of  proteins  by  enzymes  which  is 
given  in  the  latter  part  (equations  (A)  to  (D))  of  the  preceding 
section. 

As  regards  the  influence  of  alkalies  upon  the  activity  of  trypsin, 
many  of  the  facts  appear,  at  first  sight,  to  be  more  in  harmony 
with  the  view  that  the  alkali  acts  as  an  accessory  catalysor, 
since  it  is  stated  that  the  hydroxides  of  the  alkalies  and  alkaline 
earths  act  in  proportion  to  their  degree  of  dissociation  (63) 
although  divergencies  from  the  proportionality  of  the  action  to 
the  degree  of  dissociation  have  been  found  (6). 

It  has  frequently  been  pointed  out  that  the  progress  of  hy- 
drolysis of  proteins  is  accompanied  by  marked  changes  in  the 
acidity  or  alkalinity  of  the  solutions  in  which  the  hydrolysis 
occurs  (39)  (40)  (129)  (107)  (113).  This  is  to  be  attributed  to 
the  splitting  of  —  COH.N—  bonds,  in  hydrolysis,  which  do  not, 
while  bound  up  in  the  protein  molecule,  assist  in  the  neutra- 
lization of  bases  and  acids,*  but  which,  when  converted  into 
COOH—  and  H2N—  groups  may  be  presumed  to  play  the  part 
which  such  groups  ordinarily  play;  hence  any  excess  of  acid 
or  base  tends  to  disappear  during  hydrolysis.  One  might  expect 
that  this  change  in  hydrogen  or  hydroxyl  concentration  would 

*  We  have  seen  that  the  —  COH.N  —  groups  provided  by  dicarboxyl-  and 
diamino-acid  groups  are  chiefly  involved  in  the  neutralization  of  bases  and 
acids  by  proteins.  In  hydrolysis  not  only  these  bonds  are  split,  but  also  the 
—COH.N—  bonds  linking  monocarboxyl  and  monoamino-acid  groups.  It 
is  for  this  reason  that  the  combining  capacity  for  bases  of  the  products  of 
protein  hydrolysis  is  greater,  although  only  slightly  greater,  than  that  of  the 
protein  from  which  they  are  derived. 


412  CHEMICAL  DYNAMICS 

lead  to  deviation  from  regularity  in  the  relationship  between 
the  degree  and  the  time  of  hydrolysis  which  is  experimentally 
observed.  Nevertheless,  if  the  degree  of  alkalinity  be  sufficient, 
this  is  not  the  case.  Thus  in  Taylor's  experiments  upon  the 
hydrolysis  of  protamin  sulphate  the  alkalinity  of  the  system 
changed  markedly  with  the  progress  of  hydrolysis,  yet  he  ob- 
tained results  in  satisfactory  accord  with  the  monomolecular  law. 
The  reason  for  this  fact  is  very  clearly  revealed  by  the  experi- 
ments of  Robertson  and  Schmidt  (113).  We  measured  the  rate 
of  change  in  the  alkalinity  of  tryptic  digests  of  sodium  caseinate 
and  of  protamin  sulphate  (dissolved  in  dilute  alkali)  by  means 
of  the  gas  chain.  The  progressive  change  in  hydroxyl-concen- 
trations  of  these  digests,  it  was  found,  can  be  expressed  by  a 
monomolecular  formula  when  the  total  OH'  concentration  is 
greater  than  10"6,  but  thereafter,  or  when  the  OH'  concentration 
initially  is  less  than  this,  the  progressive  change  in  alkalinity 
is  no  longer  to  be  expressed  by  a  monomolecular  formula,  but 
is  represented  by  the  formula 


kt  = 


B(B- 


(where  t  is  the  time  since  the  gas-electrodes  first  came  into  equi- 
librium with  the  solution  and  B  is  the  OH'  concentration  when 
t  =  0),  which  is  characteristic  of  a  bimolecular  reaction.  For  a 
short  range  of  intermediate  alkalinities  the  order  of  the  reaction 
is  indeterminate.  These  facts  are  illustrated  by  the  tables  on 
the  following  page. 

The  point  at  which  the  order  of  the  reaction  changed  is  indi- 
cated by  an  asterisk.  It  will  be  seen  that  it  corresponds  tolerably 
closely  with  COH'  =  10"6;  and  it  will  also  be  observed  that  it  is 
the  same  for  substrates  so  very  different  as  casein  and  protamin 
sulphate.  This  indicates  that  the  change  in  the  order  of  the 
reaction  at  a  certain  OH'  concentration  depends  upon  an  alter- 
ation in  some  relationship  between  the  alkali  and  the  ferment 
and  not  upon  the  alteration  of  such  a  relationship  between 
the  substrate  and  the  ferment. 

The  most  probable  interpretation  to  be  placed  upon  these 
results  is  that  the  active  agent  in  bringing  about  the  hydrolysis 
is  not  uncombined  trypsin  but  a  compound  of  trypsin  with  the 
base  which  the  solution  contains.  The  efficiency  of  bases  in 


INFLUENCE  OF  ACID  AND  ALKALI 


413 


TABLE  I.    CASEIN 

To  AT/100  NaOH  containing  about  2  per  cent  of  casein  was  added  an 
equal  volume  of  AT/300  NaOH.  To  50  cc.  of  this  solution  were  added 
10  milligrams  of  trypsin.  Temperature  15  to  18  degrees. 


Time  in 
minutes 
after 
mixing 

t-t! 

COH/ 

z 

1 

(monomolecu- 
lar) 

1 

(bimolecular) 

51 

28.4  X10~7 

59 

"6 

27  3  X10~7 

69 

10 

26.2  X10~7 

1.1  xio-7 

180  X10~5 

154X10~6 

73 

14 

24.2  X10~7 

3.1  X10~7 

375  XIO-5 

336  XIO-6 

99 

40 

20.6  X10~7 

6.7  X10~7 

305X10-5 

296X10-6 

126 

67 

16.2  XHT7 

11.1  X10~7 

338X10-5 

376X10-6 

153 

94 

12.8  X10-7 

14.5  XIO-7 

352X10-5 

445X10-6 

197 

138 

9.3  X10-7 

18.0  X10~7 

340X10-5 

516X10-6 

225 

166 

7.6  X10-7 

19.7  XIO-7 

336X10-5 

574X10-6 

246 

187 

6.7  X10-7* 

20.6  XIO-7 

326X10-5 

600X10-6 

276 

217 

5.95X10-7 

21.35X10-7 

305X10-5 

604X10-6 

303 

244 

5.45X10-7 

21.85X10~7 

293X1Q-5 

627X10-6 

338 

279 

4.7  X10~7 

22.6  XIO-7 

275  X10~5 

635X10-6 

368 

309 

4.0  X10~7 

23.3  XIO-7 

270X10-5 

690X10-6 

408 

349 

3.7  X10-7 

23.6  XIO-7 

250X10-5 

670X10-6 

443 

384 

3.5  X10~7 

23.8  XIO-7 

232X10-5 

642  XIO-6 

470 

411 

3.3  X10~7 

24.0  X10~7 

224X10-5 

654X10-6 

547 

488 

2.8  X10-7 

24.5  X10~7 

203  XIO-5 

660  X10~6 

601 

542 

2.6  X10~7 

24.7  XIO-7 

190X10-5 

656X10-6 

647 

588 

2.5  X10~7 

24.8  X10~7 

178X10-5 

627  XIO-6 

722 

663 

2.5  X10~7 

24.8  XIO-7 

157  XIO-5 

556X10-6 

TABLE  II.  PROTAMIN  SULPHATE 

Ten  grams  of  protamin  sulphate  in  one  litre  of  solution.    Ten  milligrams 
of  trypsin  and  2.5  of  AT/10  NaOH  to  25  cc. 


Time  in 
minutes 
after 
mixing 

*-<t 

COH' 

X 

k 
(monomolecu- 
far) 

i 

(bimolecular) 

60 

28.5X10-7 

85 

0 

24.3X10-7 

0 

108 
136 
181 
241 
307 
356 
400 
453 
502 

23 

51 
96 
156 
222 
271 
315 
368 
417 

20.7X10-7 
15.0  XIO-7 
10.5X10-7 
7.0X10-7* 
5.3X10-7 
4.5X10-7 
4.0X10'7 
3.5X10-7 
3.1X10~7 

3.6X10-7 
9.3X10'7 
13.  8  XIO-7 
17.  3  XIO-7 
19.0X10-7 
19.  8  XIO-7 
20.3X10-7 
20.8X10-7 
21.2X10-7 

305  XIO-6 
411  X10~5 
380X10-5 
346X10-5 
298X10-5 
270  XIO-6 
249  XIO-5 
227  XIO-6 
213X10-5 

311X10-6 
500X10-6 
564  XIO-6 
647  X10~6 
664X10-6 
668X10-6 
664X10-6 
663  XIO-6 
676X10-6 

414  CHEMICAL  DYNAMICS 

activating  trypsin  is  thus  readily  understood.  When  the  con- 
centration of  unneutralized  base  is  in  excess  of  10  X  10~6  all  of 
the  trypsin  is  combined  with  the  base  and  in  the  form  of  the 
proteolytically  active  salt;*  but  when  the  alkalinity  falls  below 
this  limit  a  proportion  of  the  trypsin  is  no  longer  combined  with 
alkali  and  this  proportion  is  inactive.  Below  this  limit  of  OH' 
concentration,  therefore,  the  velocity-constant  of  hydrolysis 
(calculated  from  the  monomolecular  formula)  must  fall  off  in 
proportion  as  COH'  decreases,  in  other  words,  the  relation  be- 
tween COH'  and  time  will  be  expressed  by  the  bimolecular 
formula. 

This  explanation  of  our  results  is  especially  supported  by  the 
fact  that  when  the  velocity  of  hydrolysis  has  fallen  very  low, 
owing  to  decreasing  alkalinity,  the  velocity  can  be  raised  again, 
and  the  order  of  the  reaction  again  made  monomolecular,  by 
simply  increasing  the  alkalinity  of  the  solution.  This  is  shown 
in  table  III  in  wnich  the  break  indicates  that  fresh  alkali  was 
added. 

We  may  therefore  conclude  that  in  all  probability  pepsin  and 
trypsin  are  only  able  to  exert  their  proteolytic  activity  (i.e.,  act 
as  carriers  of  water)  when  they  are  present  in  their  solutions  in 
the  form  of  salts  with  acids  and  bases  respectively. 

The  above  experiments  establish  a  lower  limit  of  hydroxyl- 
concentration,  below  which  the  alkalinity  is  not  sufficient  to 
secure  the  greatest  velocity  of  transformation.  An  upper  limit, 
above  which  the  alkalinity  is  too  great  to  secure  the  greatest 
velocity  of  transformation,  is  indicated  by  the  fact  that  trypsin 
undergoes  autohydrolysis  in  solution  and  this  autohydrolysis  is 
greatly  accelerated  by  alkalies  (144)  (145);  directly,  therefore, 
the  rate  of  destruction  of  the  trypsin  by  the  excess  of  alkali 
measurably  affects  the  progress  of  the  reaction  an  upper  limit 
of  alkalinity  is  reached.  The  experiments  of  Taylor,  cited  above, 

N-  N 

establish  this  upper  limit  at  about  ^rrtr.  OH';  between  ..  ^^  ^^ 
jy  loUU  1,UUU,UUU 

and       _  •  OH',  therefore,  all  alkalinities  are  equally  favorable. 

*  Subject,  of  course,  to  dehydration  as  in  equations  (C)  and  (D)  in  the  preced- 
ing section.  In  equations  (viii)'  and  (ix)  it  is  the  value  of  F  which  is  to  be  con- 
sidered as  being  affected  by  the  alkali,  not  the  proportion  of  the  hydrated  to 
the  dehydrated  form  of  the  enzyme,  although  this  may  also  very  possibly  be 
affected  by  the  OH'  concentration,  j 


INFLUENCE  OF  ADDED  SUBSTANCES 


415 


TABLE  III 

Ten  grams  of  protamin  sulphate  in  1  litre  of  solution.    Ten  milligrams 
of  trypsin  and  2.1  cc.  of  AT/10  NaOH  to  25  cc. 


Time  in 
minutes 
after 
mixing 

,-« 

COH< 

z 

I 

(monomolecu- 
lar) 

1 

(bimolecular) 

29 

23.3  XlO"7 

38 

o 

22.4  XlO"7 

o 

63 

25 

16^25X10-7 

6.  15  X10~7 

559  XIO-5 

676X10-8 

78 

40 

13.3  X10~7 

9.1  XIO-7 

567  XIO"5 

765X10-8 

93 

55 

10.5  XIO-7 

11.9  X10~7 

601  X10~5 

920X10-8 

109 

71 

8.9  X10~7* 

13.5  X10~7 

565  XIO'5 

954X10-8 

124 

86 

7.6  X10~7 

14.8  X10~7 

545  XIO-5 

943X10-8 

132 

94 

7.0  X10~7 

15.4  X10~7 

537  X10~5 

1046X10-6 

13 

27.3  X10"7 

23 

6 

26  3  X10~7 

o 

33 

10 

24.3  XIO-7 

2.0  XIO-7 

334  XIO-5 

313X10-8 

51 

28 

20.7  XIO-7 

5.6  X10~7 

371X10-5 

367X10-8 

72 

49 

18.3  X10~7 

8.0  X10~7 

323  XIO-5 

339X10-8 

90 

67 

16.9  X10~7 

9.4  X10~7 

286X10-5 

316X10-8 

111 

88 

13.8  X10~7 

12.5  X10~7 

317  X10~5 

391X10-8 

123 

100 

12.8  X10~7 

13.5  XIO-7 

314X10-5 

401X10-6 

152 

129 

11.3  X10~7* 

15.0  X10~7 

285  XIO-5 

391X10-8 

170 

147 

10.5  X10~7 

15.8  XIO-7 

272  XIO-5 

389X10-8 

199 

176 

9.7  X10~7 

16.6  X10~7 

246X10-5 

369X10-6 

232 

209 

7.9  X10~7 

18.4  XIO-7 

250  XIO-5 

423X10-8 

251 

228 

7.6  XIO-7 

18.7  X10~7 

236X10-5 

411X10-8 

5.  The  Influence  of  Added  Substances  upon  the  Rate  of 
Protein  Hydrolysis  by  Enzymes.  —  The  influence  of  added 
substances  upon  the  rapidity  of  the  hydrolysis  of  proteins  by 
enzymes  has  been  extensively  studied,  owing  to  its  supposed 
importance  in  connection  with  the  digestion  of  protein  in  the 
alimentary  canal  (97)  (119)  (51)  (149)  (73)  (16)  (96)  (45)  (18) 
(151)  (30)  (66)  (15)  (100)  (152)  (120).  Despite  this  extended 
investigation,  however,  the  results  obtained  by  different  observers 
have  been  conflicting  in  the  extreme.  Investigations  which  I 
have  carried  out  on  the  influence  of  salts  upon  the  hydrolysis 
of  caseinates  by  trypsin  indicate  the  probable  source  of  the  dis- 
crepancies between  the  results  of  different  observers  (107).  I 
find  that  the  order  of  efficiency  of  salts  in  accelerating  the  hy- 
drolysis of  casein  by  trypsin  is  the  order  of  their  efficiency  in 
accelerating  the  solution  of  casein  by  alkalies.  If  this  rule  holds 
good  for  other  proteins,  then  one  might  anticipate  that  the 
influence  of  salts  upon  their  hydrolysis  by  enzymes  would  vary 


416  CHEMICAL  DYNAMICS 

somewhat  with  the  nature  of  the  protein  or  protein  salt  employed 
as  substrate. 

The  effect  of  salts  in  accelerating  the  hydrolysis  of  casein  by 
trypsin  is,  in  some  instances,  very  marked.  Thus  in  a  solution 
of  "neutral"  calcium  caseinate  containing  143  milligrams  of 
casein  in  200  cc.  plus  1  cc.  of  a  0.025  per  cent  solution  of  Gruebler's 
trypsin  at  36.5  degrees  the  velocity-constant  of  hydrolysis  was 
11  (calculated,  in  arbitrary  units,  from  the  monomolecular  for- 
mula) while,  upon  the  addition  of  0.05  N  NaCl  the  value  of  the 
constant  rises  to  44  (in  the  same  units). 

The  influence  of  the  concentration  of  the  added  NaCl  upon 
the  acceleration  of  the  tryptic  hydrolysis  of  caseinates  which 
it  brings  about  is  of  great  interest,  since,  so  far  as  my  observa- 
tions have  extended,  it  is  of  the  same  nature  as  its  influence 
upon  the  degree  of  swelling  which  gelatin  plates  undergo  when 
immersed  in  water.  It  will  be  recollected  (Cf.  Chap.  XII)  that 
when  gelatin  plates  are  immersed  in  solutions  of  NaCl  of  vary- 
ing concentration,  the  degree  of  swelling  which  they  attain  after 
a  given  period  does  not  increase  continuously  with  the  concen- 
tration of  the  solution  but  exhibits  marked  maxima  and  minima 
at  definite  concentrations  (94).  Exactly  similar  phenomena 
occur  when  NaCl  in  varying  concentration  is  added  to  a  tryptic 
digest  of  casein.  The  acceleration  in  hydrolysis  which  is  pro- 
duced by  the  added  NaCl  does  not  continuously  increase  with 
increasing  concentration  of  the  NaCl,  but  exhibits  marked  max- 
ima and  minima. 

6.  The  Action  of  the  Coagulating  Ferments,  Rennet  and 
Thrombin.  —  As  is  well  known,  rennet  occurs,  together  with 
pepsin,  in  the  gastric  juice  and  in  the  juices  of  many  plants  and 
brings  about  the  clotting  of  milk  through  the  transformation 
of  casein  into  paracasein.  Thrombin  occurs  in  some  part  of  the 
circulating  blood,  possibly  the  leucocytes,  and  brings  about  the 
coagulation  of  blood  through  the  transformation  of  fibrinogen 
into  fibrin.  The  modes  of  action  of  these  ferments  have  been 
the  subject  of  very  many  investigations  (102)  (103)  (31)  (84) 
(75)  (61),  which  cannot  be  dwelt  upon  in  detail  here.  In  brief, 
however,  it  may  be  stated  that  rennet  converts  casein  into 
paracasein,  whether  calcium  salts  are  present  or  not,  and  the 
paracasein  having  been  formed  (which  is  soluble  in  the  absence 
of  salts  of  the  alkaline  earths),  it  is  coagulated  (or  precipitated; 


COAGULATING  FERMENTS 


417 


Cf.  Chap.  VI)  by  calcium  salts  or  other  salts  of  the  alkaline 
earths.  The  part  played  by  calcium  in  forwarding  the  coagu- 
lation of  blood  is  very  different;  here  the  dependency  is  one 
of  the  ferment  (or  a  pro-ferment),  and  not  of  the  coagulation 
of  the  substrate,  upon  the  presence  of  calcium  salts;  it  would 
appear  that  calcium  salts  activate  a  pro-thrombin,  converting  it 
into  the  ferment  thrombin,  which  will  then  induce  coagulation 
whether  calcium  salts  be  present  or  not.  The  function  of  cal- 
cium in  these  two  processes  is  therefore  very  different. 

Fuld  (32),  Gerber  (37)  and  Madsen  (77)  have  shown  that  for 
the  coagulation  of  milk  by  rennet  F  X  t  —  constant;  i.e.,  the 
velocity  of  coagulation  is  proportional  to  the  concentration  of 
ferment.  Regarding  the  influence  of  calcium  salts,  very  inter- 
esting results  have  been  obtained  by  Reichel  and  Spiro  (104). 
These  observers  added  to  8  cc.  of  milk  different  quantities  (R) 
of  rennet  solution  and  different  amounts  (p)  of  calcium  chloride, 
and  diluted  with  water  to  a  total  volume  of  100  cc.  They  then 
measured  the  time  required  for  coagulation  at  a  constant  tem- 
perature. The  following  were  their  results : 


CaCh  in 

Time  of  coagulation 

Constant  =  (p  +  0.6)  t 

P 

R  =  1  cc. 

R  =  0.5  cc. 

R  =  0.25cc. 

R  =  lcc. 

R  =  0.5  cc. 

R  =  0.25  cc. 

0.00 

95.0 

48.0 

24.0 

57.0 

28.8 

14.4 

0.05 

88.6 

45.6 

23.0 

57.6 

29.6 

15.0 

0.1 

79.0 

41.6 

22.0 

55.3 

29.1 

15.4 

0.2 

66.4 

36.0 

19.0 

53.1 

28.8 

15.2 

0.5 

48.0 

26.4 

14.0 

52.8 

29.0 

15.4 

1.0 

30.0 

18.2 

10.6 

48.0 

29.1 

17.0 

2.0 

17.0 

11.0 

6.8 

44.2 

28.6 

17.7 

5.0 

10.0 

7.4 

5.4 

56.0 

41.4 

30.2 

10.0 

13.0 

9.2 

6.2 

137.8 

97.5 

65.7 

20.0 

22.0 

15.0 

8.6 

453.2 

309.0 

177.2 

It  is  evident  that  the  product  (p  +  0.6)  t  is  constant  for  each 
value  of  R,  provided  p  does  not  exceed  2  per  cent.  The  velocity 
is  also  proportional  to  R}  so  that  the  complete  relationship  is 
R  (p  +  0.6)  t  =  constant.  The  quantity  0.6  which  must  be  added 
to  p  in  the  above  equation  is  to  be  regarded  as  the  (percentage) 
concentration  of  calcium  ions  present  already  in  milk,  before 
any  calcium  chloride  is  added.  According  to  Reichel  and  Spiro 
this  indicates  that  only  about  15  per  cent  of  the  calcium  in  milk 


418  CHEMICAL  DYNAMICS 

is  ionic;  this  obviously  corresponds  very  well  with  the  statement 
in  Chap.  VIII  and  elsewhere  in  this  work  that  the  calcium  bound 
in  calcium  caseinate  is  not  electrolytically, dissociated  as  such. 

In  the  opinion  of  Nencki  and  Sieber  (86),  Schoumov-Simanovski 
(123),  Pavlov  and  Parastschuk  (95),  Savjallov  (118),  Gevin  (38), 
Migay  and  Savitsch  (83),  Van  Dam  (139)  (140)  (141),  Savitsch 
(117),  Herzog  (57),  Funk  and  Niemann  (34),  Laqueur  (71)  (72) 
and  Bosworth  (12)  (13),  rennet  and  pepsin  are  identical  sub- 
stances, the  transformation  of  casein  into  paracasein  being  the 
first  step  in  hydrolysis  or  else  a  side-hydrolysis  (135).  On  the 
other  hand,  Hammarsten  (43)  (44),  Hedin  (50),  Rakoczy  (101) 
and  others  are  of  the  opinion  that  rennet  and  pepsin  are  dis- 
tinguishable from  one  another.  The  evidence  for  the  identity 
of  the  two  enzymes  lies  in  the  fact  that  in  no  way,  either  quanti- 
tatively or  qualitatively,  can  the  activity  of  rennet  be  separated 
from  that  of  pepsin,  statements  to  the  contrary  effect  (4)  (52) 
(135)  (62)  (122)  (43)  having  been  disproved  or  else  shown  to 
rest  upon  experimental  data  obtained  under  conditions  neces- 
sarily involving  inhibition,  or  apparent  inhibition,  of  the  one 
form  of  action  or  of  the  other  (Cf.  especially  Van  Dam,  (139) 
(140)  (141)).  This  is  very  well  illustrated  by  the  following 
experiment  of  Morgenroth  (85).  If  mixtures  of  calcium  caseinate 
and  rennet  are  kept  at  low  temperatures  no  coagulation  occurs, 
but  the  digestion  process  does  occur;  after  these  mixtures  have 
been  held  for  some  time  at  the  low  temperature,  if  they  are  heated 
to  20  degrees  they  coagulate  instantly.  Thus  the  process  which 
underlies  the  clotting  takes  place  at  the  lower  temperature  but 
cannot  be  evidenced  by  actual  clotting  until  the  temperature  has 
risen.  It  has  repeatedly  been  pointed  out  (105)  (106)  (72)  that 
the  difference  between  the  behavior  of  paracasein  towards  calcium 
salts  and  that  of  casein  is  essentially  this,  that  calcium  para- 
caseinate  is  coagulated  by  calcium  salts  at  lower  temperatures 
than  calcium  caseinate. 

Now  it  has  been  shown  by  Van  Slyke  and  Hart  (143),  Geake 
(36)  and  Van  Slyke  and  Bosworth  (142)  that  the  paracasein 
freed  from  its  combination  with  calcium  is  analytically  indis- 
tinguishable from  casein,  and  Van  Slyke  and  Bosworth  have 
shown  that  the  minimal  combining-capacity  of  paracasein  for 
bases  is  exactly  double  that  of  casein.  These  facts  admit  of 
only  one  interpretation,  namely,  that  casein  is  hydrolytically 


INFLUENCE  OF  TEMPERATURE  419 

split  by  rennet  into  two  equal  parts.  The  correctness  of  this 
interpretation  has  been  completely  confirmed  by  Bosworth  (12) 
who  has  shown  that  in  the  first  place  no  substance  other  than 
paracasein  results  from  the  action  of  rennet  upon  casein,  while, 
in  the  second  place,  no  matter  what  proteolytic  enzyme  (rennet 
or  trypsin)  be  employed  to  split  the  casein,  the  first  step  in 
hydrolysis  is  the  production  of  paracasein  (13). 

As  regards  the  kinetics  of  the  action  of  fibrin-ferment  (throm- 
bin)  Fuld  (33)  finds  that  the  Schtitz  rule  (velocity  proportional 
to  the  square  root  of  the  ferment-mass)  very  nearly  holds  good, 
the  actual  relation  between  the  velocity  (v)  of  coagulation  and 
the  mass  of  fibrin-ferment  (E)  being: 


C.  J.  Martin  finds  that  for  the  coagulation  of  blood  by  throm- 
bin  F  X  t  =  constant  (78). 

Fibrin-ferment  undergoes  marked  exhaustion  during  the 
process  of  coagulation  (84)  (60) . 

7.  The  Influence  of  Temperature  upon  the  Velocity  of  Protein 
Hydrolysis.  —  The  influence  of  temperature  upon  the  hydrolysis 
of  gelatin  by  trypsin  has  been  carefully  studied  by  Madsen  and 
Walbaum  (cited  after  Arrhenius  (3)).  The  results  are  in  good 
agreement  with  the  formula: 


where  e  is  the  base  of  the  Napierian  logarithms,  Vi  and  v0  are  the 
velocities  corresponding  to  absolute  temperatures  TI  and  T0  and 
IJL  is  a  constant,  in  this  instance  10,570.  This  is  the  law  which 
is  characteristic  of  the  influence  of  temperature  upon  other 
chemical  reactions,  and  its  possible  significance,  when  it  is  found 
applicable  to  protein  systems,  has  been  commented  upon  in 
Chap.  VII. 

The  influence  of  temperature  upon  the  rate  of  digestion  of 
thymol-gelatin  by  pepsin  is  also  capable  of  representation  by  the 
above  formula  (Arrhenius,  loc.  cit.,  p.  71).  In  this  case  /z  =  10,750. 
The  velocity  both  of  pepsin  and  trypsin-digestion  is,  therefore, 
doubled  by  a  rise  in  temperature  from  20  to  30  degrees. 

Using  different  substrates  it  is  found  that,  as  might  be  expected, 


420  CHEMICAL  DYNAMICS 

the  value  of  p  for  hydrolysis  by  a  given  enzyme  varies  with  the 
nature  of  the  substrate.  (Cf.  table  in  Arrhenius'  work,  loc.  cit., 
pp.  99-98.) 

The  influence  of  temperature  upon  the  coagulative  power  of 
rennet  has  been  studied  by  Fuld  (32) ;  here  also  the  van't  Hoff- 
Arrhenius'  rule  holds  good;  /*  is  20,650. 

The  interesting  observation  has  been  made  by  Cesana  (17)  in 
studying  the  influence  of  temperature  upon  the  ultramicroscopic 
appearance  of  trypsin  solutions,  that  the  optimum  temperature 
for  its  proteolytic  activity  corresponds  with  that  at  which  there 
is  a  minimum  of  visible  granules  in  the  ultramicroscopic  field 
and  a  maximum  dispersion  of  the  trypsin  leading  to  a  maximum 
frequency  of  collisions  between  trypsin  particles  or  molecules 
and  the  molecules  of  the  substrate. 


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CHAPTER  XVII 
THE  ENZYMATIC  SYNTHESIS  OF  PROTEINS 

1.  The  Reversion  of  the  Hydrolysis  of  Proteins  by  Pepsin  and 
Trypsin.  —  In  "typical"  catalysed  reactions  (Cf.  previous  Chap- 
ter, section  1),  since  the  equilibrium  of  the  system  is  not  shifted 
by  the  catalysor  and  the  point  of  equilibrium  is  determined  by 
the  ratio  of  the  velocity-constants  of  the  forward  and  the  opposed 
reactions,  both  velocity-constants  must  be  multiplied  to  an  equal 
degree  by  the  catalysor;  in  other  words,  if  under  given  conditions 
a  point  of  equilibrium  exists  at  which  an  appreciable  proportion 
of  the  substrate  remains  unaltered,  then,  under  such  conditions, 
the  pure  products  (unmixed  with  substrate)  are  not  in  equilibrium 
and  must  be  tending  to  restore  the  substrate,  and  this  tendency 
must  be  increased  by  the  presence  of  the  catalysor  in  question. 

As  this  fact  became  generally  appreciated,  following  its  ex- 
perimental verification  in  a  large  number  of  non-fermentative 
catalytic  reactions,  and  as  the  belief  gained  ground  that  the 
ferments  were  to  be  regarded  as  "typical"  catalysors,  it  was 
anticipated  that  fermentative  reversions  might  be  accomplished, 
that  is  the  synthesis  of  substances  which  are  normally  hydrolysed 
(or  otherwise  altered)  by  enzymes,  through  acting  upon  the 
products  of  hydrolysis  with  the  same  enzymes.  This  theoretical 
anticipation  was  first  experimentally  realized  by  Croft  Hill  (31) 
(32)  in  the  fermentative  synthesis  of  isomaltose  (16)  (2)  (3) 
from  glucose,  and  has  since  been  realized  in  the  fermentative 
synthesis  of  polysaccharides,  fats,  and  proteins  by  various  investi- 
gators  (7)  (34)  (26)  (8)  (66)  (67)  (69)  (70)  (47)  (48)  (9)  (51)  (52). 

In  order  to  make  clear  the  conditions  under  which  such  re- 
versions occur  it  will  be  well  to  consider  in  some  detail  the  funda- 
mental experiment  performed  by  Croft  Hill. 

The  hydrolysis  of  maltose  (or  of  isomaltose)  is  represented 
by  the  equation: 

(A)  Ci2H220n  +  H20  -» 2  C6H1206; 

the  reversion  is  represented  by  the  equation : 

(B)  2  C6H1206  -»  CuH^On  -|-  H20. 

425 


426  CHEMICAL  DYNAMICS 

The  condition  of  "  balance"  at  equilibrium  is  therefore  rep- 
resented by  the  equation: 

(C)  Ci2H22On  +  H2O  <±  2  C6H1206. 


If  mi  be  the  mass  of  maltose,  w2  that  of  glucose  and  W  that  of 
water,  then  at  equilibrium  we  shall  have,  in  accordance  with  the 
mass-law  : 

miW  =  j*mf,  (i) 

in  which  k  and  &2  are  the  velocity-constants  of  the  forward  and 
the  reverse  reactions,  respectively.  If  maltase  is  indeed  a 
"typical"  catalysor,  then  we  must  assume  that  although  ki 
and  kz  are  each  multiplied  by  the  presence  of  the  catalysor  their 
ratio  remains  unaffected  and,  consequently,  the  relative  pro- 
portions between  mi  and  mz  and  W  at  equilibrium  are  not  affected 
by  the  presence  of  maltase.* 

Now  the  value  of  the  ratio  -^  for  this  reaction  is  such  that  in 

ki 

dilute  solutions  (i.e.,  when  W  in  equation  (i)  is  large)  the  point 
of  equilibrium  in  equation  (C)  lies  so  far  over  towards  the  right 
that  hydrolysis  of  the  maltose,  at  the  end  of  the  reaction,  is 
practically  complete,  no  appreciable  quantity  of  unhydrolysed 
maltose  remaining  in  the  system.  This  being  the  case  we  cannot 
expect,  through  the  agency  of  any  "typical"  catalysor,  to  secure 
the  synthesis  of  maltose  in  a  dilute  solution  of  glucose.  A  con- 
siderable increase  in  the  concentration  of  the  system,  however, 
although  it  does  not  bring  about  any  appreciable  alteration 
either  in  ki  or  in  &2,  nevertheless  alters  the  proportion  of  maltose 
to  glucose  at  equilibrium  very  profoundly.  The  reason  for  this 
is  apparent  upon  inspection  of  equation  (i).  Increasing  the 
concentration  of  the  system  is  equivalent  to  reducing  the  mag- 
nitude of  W  and  hence  inducing  a  compensating  increase  in  mi 
(the  mass  of  maltose)  at  equilibrium.  In  a  concentrated  system 
we  find,  accordingly,  that  the  hydrolysis  of  maltose  is  never 
complete,  whether  maltase  or  any  other  catalysor  be  employed 
to  accelerate  the  process.  A  concentrated  solution  of  glucose  is 
therefore  not  in  a  condition  of  equilibrium  until  a  proportion 
of  maltose  has  been  restored  to  the  system  by  the  occurrence  of 

*  It  should  be  clearly  borne  in  mind  that  the  verity  of  this  statement  has 
never  been  proven. 


REVERSION  OF  HYDROLYSIS  427 

reaction  (B) .  In  the  absence  of  any  catalysors  this  reaction  takes 
place  very  slowly,  but  if  any  of  the  catalysors  be  present  which 
are  capable  of  accelerating  the  hydrolysis  of  maltose,  for  example 
maltase,  then  the  synthesis  of  maltose  or  of  isomaltose  occurs 
with  measurable  rapidity.  It  was  by  adding  maltase  to  con- 
centrated glucose  that  Croft  Hill  obtained  a  fermentative  synthesis 
of  isomaltose. 

In  satisfactory  accord  with  the  above  account  of  the  chemical 
mechanics  of  fermentative  reversions,  the  majority  of  observers 
have  found  that  reversion  of  a  fermentative  hydrolysis  is  only 
attainable  when  the  enzyme  is  made  to  act  upon  the  concentrated 
products  of  the  hydrolysis.  Nevertheless  reversion  does  not 
always  occur  when  it  might  be  expected  to  occur;  thus  Bradley 
(9)  has  shown  that  in  the  presence  of  50  per  cent  of  water  no 
appreciable  synthesis  of  triolein  from  glycerin  and  oleic  acid 
can  be  brought  about  through  the  agency  of  lipase,  although 
in  the  presence  of  50  per  cent  of  water  an  appreciable  proportion 
of  triolein  remains  unhydrolysed.  The  converse  is  also  true, 
reversion  occurs  when  it  might  not  be  anticipated.  Thus  I  find 
(53)  that  it  is  possible  to  completely  hydrolyse  paranuclein  to 
products  which  have  lost  its  characteristic  properties  and  yet, 
without  any  previous  concentration,  to  bring  about,  through 
simply  altering  the  temperature  and  the  enzyme  concentration, 
a  fermentative  synthesis  of  this  substance.  These  apparent 
anomalies  arise  from  the  fact  that  these  ferments  do  not  belong 
to  that  restricted  class  of  catalysors  which,  as  explained  in  sec- 
tion 1  of  the  previous  chapter,  has  come  to  be  regarded  as  typical, 
but  to  what  Stieglitz  has  shown  to  be  the  more  general  class  of 
catalysors,  namely  those  which  appreciably  participate  in  the 
reactions  which  they  accelerate  and  therefore  appreciably  disturb 
the  final  equilibrium.  The  significance  of  these  phenomena,  in  so 
far  as  the  proteolytic  enzymes  are  concerned,  will  be  more 
fully  discussed  below. 

The  fermentative  synthesis  of  protamin  through  the  action 
of  trypsin  upon  the  concentrated  products  of  its  hydrolysis  has 
been  accomplished  by  Taylor  (69)  (70)  and  I  have  described  the 
fermentative  synthesis  of  paranuclein  (51)  (52)  through  the 
action  of  pepsin  upon  the  products  of  its  hydrolysis. 

In  Taylor's  experiments  the  mixed  products  of  the  tryptic 
digestion  of  protamin  sulphate  were  converted  into  carbonates 


428  CHEMICAL  DYNAMICS 

and  then  (the  ferment  having  been  destroyed  by  boiling)  evapo- 
rated until  precipitation  (at  room  temperatures)  just  began. 
The  solution  which  was  thus  prepared  contained  the  products  of 
the  hydrolysis  of  400  grams  of  protamin  and  was  free  from  un- 
hydrolysed  protamin,  since  the  original  solution  (before  evapo- 
ration) was  miscible  with  five  volumes  of  acidulated  alcohol 
without  any  resulting  opacity.  To  this  solution  were  then  added 
300  cc.  of  a  glycerin  extract  of  the  livers  of  the  large  soft-shelled 
California  clam  (Schizothcerus  nuttalli),  which  contain  a  proteo- 
lytic  ferment  of  the  trypsin  type  which  is  very  resistant  to  auto- 
destruction  and  therefore  especially  adapted  for  employment  in 
experiments  of  long  duration.  After  the  addition  of  this  trypsin 
the  solution  was  still  miscible  with  alcohol  without  cloudiness. 
Excess  of  toluol  was  then  added,  and  the  flask  (containing  over 
4  litres)  was  set  aside  at  room  temperature. 

The  mixture  gradually  became  opalescent,  then  cloudy,  and, 
finally,  after  the  lapse  of  some  months,  a  distinct  precipitate 
had  formed.  After  five  months  the  flask  was  opened,  the  con- 
tents were  heated  to  boiling  to  destroy  the  ferment,  acidulated 
with  sulphuric  acid,  which  dissolved  the  precipitate,  and  filtered. 
Then  four  volumes  of  absolute  alcohol  were  added,  which  resulted 
in  the  throwing  down  of  a  heavy  white  precipitate,  which,  after 
purification,  was  analysed.  In  all,  about  2  grams  were  obtained. 

The  percentage  formula  of  the  protamin  sulphate  employed 
(salmin  sulphate  prepared  from  the  spermatozoa  of  Roccus 
lineatus)  had  previously  been  determined  by  Taylor  and  found 
to  be: 

H2S04. 


The  analysis  of  the  synthetical  preparation  yielded  the  following 
results  : 


Calculated  for 
C3oH«oN17O6.2  H2SO<, 
per  cent 

Found, 
per  cent 

c  =  . 

37.85 

37  68 

H  =  

6.72 

6  89 

N  - 

25  13 

24  90 

H2SO4  = 

20  60 

20  68 

The  substance  was  digestible  by  trypsin  and  not  digestible  by 
pepsin.    Taylor  concludes  that  it  was  salmin.     A  concentrated 


REVERSION  OF  HYDROLYSIS  429 

solution  of  the  products  of  the  hydrolysis  of  protamin,  to  which 
no  ferment  had  been  added,  yielded  no  trace  of  protamin  after 
standing  for  several  months. 

My  experiments  were  conducted  as  follows:  Four  hundred  cc. 
of  N/W  potassium  hydroxide  were  neutralized  (or  rendered 
faintly  acid)  to  litmus  by  the  addition  of  casein  and  the  solu- 
tion was  subjected  to  the  action  of  pepsin  for  a  considerable 
period,  fresh  pepsin  Q  g.  per  litre)  being  added  from  time  to  time, 
at  40°  C.  The  digest  at  the  end  of  this  period,  contained  no 
casein,  but  a  considerable  quantity  of  paranuclein  had  been 
precipitated  and  had  not  undergone  hydrolysis.  The  digest  was 
then  heated  to  100  degrees  for  about  10  or  15  minutes  to  destroy 
the  ferment,  and  filtered  while  hot.  The  solution  was  then 
evaporated  on  a  water-bath  to  about  70  cc.  (the  total  concen- 
tration of  the  system  having  been  thus  multiplied  by  about  6). 
This  concentrated  solution  of  the  products  of  the  peptic  digestion 
of  casein  (and  its  first  product  of  hydrolysis,  paranuclein)  is  a 
clear  brown  syrup  which  is  strongly  acid  and  gives  no  precipitate 
or  opalescence  upon  the  addition  of  acetic  acid,  or  upon  the 
addition  of  acetic  acid  in  excess  after  previously  rendering  the 
solution  alkaline  by  the  addition  of  KOH  or  NaOH.  Both  casein 
and  paranuclein  are  therefore  absent  from  it.  To  70  cc.  of  this 
solution  were  added  30  cc.  of  a  concentrated  (approximately 
10  per  cent)  solution  of  Gruebler's  pepsin  puriss.  sice,  which  had 
previously  been  filtered  through  rapid-filtering  paper.  The 
mixture  of  the  two  solutions  is  a  clear  brown  syrupy  fluid  which 
yields  no  trace  of  a  precipitate  with  acetic  acid  either  before  or 
after  neutralization  with  alkali.  The  mixture  was  set  aside  at 
40°  C.  in  the  presence  of  excess  of  toluol  to  prevent  bacterial 
infection.  Within  two  hours  a  thick  white  precipitate  had  formed 
in  the  solution;  after  48  hours  the  solution  was  filtered  and  the 
precipitate  thus  collected  on  the  filter  was  dissolved  in  a  minimal 
amount  of  sodium  hydroxide  and  the  filter  so  arranged  that  the 
alkaline  solution  dropped  directly  into  water  acidified  with  acetic 
acid.  The  precipitate  thus  obtained  was  reprecipitated  twice 
and  was  washed  by  decantation  several  times,  employing  several 
litres  of  water  at  each  decantation.  Finally  it  was  collected  on 
a  hardened  filter,  washed  with  several  litres  of  alcohol  and  ether, 
and  dried,  first  over  CaCk  and  then  over  H2S04  at  60°  C.  In 
this  way  1.02  grams  of  a  friable  greyish-white  hygroscopic  powder 


430  CHEMICAL  DYNAMICS 

were  obtained.  This  substance  gave  every  indication  of  being 
identical  with  paranuclein,  the  first  product  of  the  peptic  hy- 
drolysis of  casein.  Accordingly,  for  purposes  of  comparison, 
paranuclein  was  prepared  by  partly  digesting  sodium  caseinate 
with  pepsin,  filtering  off  the  precipitate,  dissolving  in  sodium 
hydroxide  and  precipitating  with  acetic  acid.  The  paranuclein 
was  reprecipitated  twice,  washed  with  large  volumes  of  water, 
alcohol,  and  ether,  and  dried  over  CaCl2  and  later  over  H2S04 
at  60  degrees.  A  powder  exactly  similar  in  appearance  to  the 
synthesized  substance  was  thus  obtained. 

The  synthesized  substance  and  the  paranuclein  were  both 
analysed  for  phosphorus  by  Neumann's  method,  with  the  follow- 
ing results : 

Synthetic  Substance  Paranuclein 

P2O5  =  1.61  per  cent  P2O5  =  4.18  per  cent 

Previous  observers  agree  in  stating  that  the  percentage  com- 
position of  paranuclein  varies  very  greatly  with  the  circumstances 
under  which  it  is  prepared,  the  percentages  of  phosphorus  which 
have  been  found  in  various  preparations  varying  from  0.88  to 
6.86  (43).  This  fact  leads  us  to  suspect  that  the  substance 
which  has  been  termed  paranuclein  is,  in  reality,  a  mixture  of 
at  least  two  substances  and  the  hypothesis  which  has  suggested 
itself  to  me  is  that  during  the  hydrolysis  of  casein  by  pepsin  a 
substance  of  high  phosphorus  content,  insoluble  in  dilute  acids, 
is  first  formed;  that  this  substance  splits  off  a  soluble  phosphorus- 
containing  moiety,  leaving  another  insoluble  substance  of  lower 
phosphorus  content,  and  that  this  second  substance  is  in  its  turn 
attacked  and  split  up  into  soluble  substances.  That  this  expla- 
nation is  probably  the  correct  one,  although,  of  course,  several 
such  steps  may  possibly  be  involved,  is  shown  by  the  following 
experiment. 

One  gram  of  the  paranuclein  containing  4.18  per  cent  of  P2O5 
was  dissolved  in  400  cc.  of  0.045  N  Ca(OH)2  and  the  mixture 
was  allowed  to  stand  at  40°  C.  for  12  hours;  acetic  acid  was  then 
added  in  excess  and  the  precipitate  purified,  washed  and  dried 
in  the  manner  described  above.  This  substance,  which  we  may 
designate  paranuclein  A,  was  analysed  for  phosphorus  and  found 
to  contain  1.51  per  cent  of  P205,  thus  agreeing  closely  with  the 
synthetic  substance.  Only  a  little  over  0.2  gram  of  this  sub- 


REVERSION  OF  HYDROLYSIS  431 

stance  was  obtained  from  the  gram  of  paranuclein  originally 
dissolved  in  the  lime-water. 

The  splitting  off  of  phosphorus  from  paranuclein  in  alkaline  solu- 
tion has  been  commented  on  by  other  observers  (61). 

The  substance  which  is  obtained  synthetically,  as  described 
above,  resembles  paranuclein  A  very  closely  both  in  general  prop- 
erties and  in  its  percentage-content  of  P20&.  The  synthesized 
substance  is  insoluble  in  dilute  weak  acids,  readily  soluble  in 
dilute  alkali,  precipitates  protamin  from  a  1  per  cent  solution  of 
the  sulphate  at  a  reaction  just  alkaline  to  phenolphthalein  (at 
which  reaction  both  the  protamin  and  the  paranuclein  remain 
in  stable  solution  when  not  mixed),  and  a  2  per  cent  solution  in 
N/ 10  sodium  hydroxide  is  precipitated  by  m/10  ferric  ammo- 
nium sulphate;  these  being  all  well-known  properties  of  para- 
nuclein (61)  (42). 

The  synthesized  substance  also  resembles  my  preparation  of 
paranuclein  A  in  the  following  properties;  in  approximately 
2  per  cent  solution  in  N / 100  NaOH  it  gives  the  xanthoproteic, 
Millon's,  Adamkiewicz  and  the  biuret  (violet)  reactions;  it  is 
precipitated  by  cupric  chloride  (1  vol.  of  N/l  to  100)  and  by 
zinc  chloride,  but  not  by  mercuric  chloride  (5  vols.;  of  JV/10) 
it  is  precipitated  by  picric  and  tannic  acids,  but  the  precipitate 
redissolves  on  rendering  the  solution  alkaline;  it  is  not  precipi- 
tated by  the  addition  of  five  volumes  of  absolute  alcohol;  and 
the  precipitate  which  is  at  first  produced  by  the  addition  of  acetic 
acid  is  soluble  in  considerable  excess  of  the  reagent. 

The  change  in  the  refractive  index  of  JV/50  KOH  which  is 
brought  about  by  the  introduction  of  one  per  cent  of  the  synthetic 
substance  is  identical  with  that  which  is  brought  about  by  the 
introduction  of  one  gram  of  paranuclein  or  paranuclein  A  (54), 
namely,  0.00140  for  sodium  light. 

If  no  pepsin  be  added  to  the  concentrated  solution  of  the 
products  of  the  peptic  digestion  of  casein,  prepared  as  described 
above,  the  solution,  after  keeping  for  over  a  year  at  room-tem- 
peratures or  for  several  months  at  40°  C.  remains  perfectly  clear 
and  homogeneous  and  gives  no  tests  for  paranuclein  or  for  casein. 
A  10  per  cent  filtered  solution  of  Gruebler's  pepsin,  on  standing 
for  many  months  at  40  degrees,  also  remains  perfectly  clear  and 
homogeneous.  Yet  these  two  solutions,  when  mixed  in  the 
proportion  of  one  volume  of  ferment-solution  to  five  volumes  of 


432  CHEMICAL  DYNAMICS 

the  solution  of  the  casein-products,  give  a  voluminous  precipitate 
which  remains  permanent  for  many  weeks. 

The  fact  that  paranuclein  A,  rather  than  paranuclein  or  casein, 
is  produced  in  the  reversion  of  protein  hydrolysis  which  pre- 
sumably occurs  in  the  experiments  described  above,  is  probably 
to  be  interpreted  as  follows : 

In  the  initial  stages  of  the  hydrolysis  of  casein,  paranuclein 
is  produced,  part  of  which  undergoes  further  hydrolysis  and 
part  of  which  escapes  further  hydrolysis  owing  to  the  fact  that 
it  is  thrown  out  of  the  sphere  of  action  of  the  enzymes  by  pre- 
cipitation (59)  (60)  (61).  The  proportion  of  the  paranuclein 
which  undergoes  hydrolysis  passes  through  the  intermediate 
stage  paranuclein  A  and  then  to  the  stage  of  proteoses  and 
peptones.  Only  that  proportion  of  the  paranuclein  which  under- 
goes this  further  hydrolysis,  therefore,  yields  to  the  filtered  and 
concentrated  digest  all  of  the  substances  which  are  necessary 
for  resynthesis.  In  event  of  synthesis  occurring  in  a  mixture 
such  as  that  described  above,  the  first  substance  which  resulted 
which  is  insoluble  in  solutions  of  weak  acids  would  necessarily 
be  thrown  out  of  the  sphere  of  action  and  the  reaction  would 
terminate  at  this  point.  Hence,  from  what  has  been  said  above, 
it  is  evident  that  were  a  member  of  the  paranuclein  group  pro- 
duced in  these  mixtures  it  would  be  paranuclein  A,  rather  than 
one  of  the  other  members  of  the  group  of  higher  phosphorus- 
content. 

In  the  above  experiments,  in  order  to  secure  synthesis  of  the 
paranuclein,  the  products  of  its  hydrolysis  had  to  be  concen- 
trated to  a  considerable  degree.  From  what  has  been  said  in 
the  beginning  of  this  section  the  rationale  of  this  procedure  will 
be  evident.  Later  experiments,  however  (53),  showed  that 
previous  concentration  of  the  products  of  the  complete  peptic 
hydrolysis  of  paranuclein  is  not  necessary  if  the  synthesis  be 
carried  out  in  the  presence  of  a  considerable  excess  of  pepsin 
and  at  a  much  higher  temperature,  namely  from  60  to  70  de- 
grees. 

I  made  up  the  following  mixtures  in  duplicate,  having  first 
ascertained  that  the  unconcentrated  products  of  the  peptic 
hydrolysis  of  AT/50  alkali  caseinates  and  10  per  cent  pepsin 
(Gruebler's  puriss.  sice.)  can  be  kept  separately  for  weeks  at 
65  degrees  without  a  trace  of  precipitate  forming  in  either  solution. 


REVERSION  OF  HYDROLYSIS  433 

(a)  10  cc.  of  unconcentrated  products  +  0.5  cc.  of  15  per  cent  pepsin. 

(6)  10  cc.  of  unconcentrated  products  +  1.0  cc.  of  15  per  cent  pepsin. 

(c)  10  cc.  of  unconcentrated  products  +  1.5  cc.  of  15  per  cent  pepsin. 

(d)  10  cc.  of  unconcentrated  products  +  2.0  cc.  of  15  per  cent  pepsin. 

(e)  10  cc.  of  unconcentrated  products  +  3.0  cc.  of  15  per  cent  pepsin. 

The  one  set  was  kept  at  65  degrees,  while  the  other  was  kept 
at  36  degrees,  both  in  tightly  stoppered  vessels  containing  excess 
of  toluol.  After  24  hours  there  was  no  sign  of  any  precipitate 
or  opalescence  in  the  mixtures  which  had  been  kept  at  36  degrees, 
while  in  the  duplicate  set,  which  had  been  kept  at  65  degrees, 
(e)  contained  a  heavy  precipitate  which  left  the  supernatant 
fluid  clear,  (c)  and  (d)  contained,  also,  heavy  precipitates  which, 
however,  left  the  supernatant  fluid  strongly  opalescent,  and  (a) 
and  (b)  both  contained  slight  precipitates.  After  24  hours  more, 
no  change  had  occurred  in  any  of  the  solutions  and  those  which 
had  been  kept  at  65  degrees  were  now  returned  to  36  degrees. 
After  a  lapse  of  three  weeks  no  trace  of  precipitate  had  appeared 
in  any  of  those  solutions  which  had  been  at  36  degrees  throughout, 
while  no  further  change  had  occurred  in  those  which  had  been 
kept  at  65  degrees  for  48  hours. 

The  probable  identity  of  the  precipitate  which  is  thus  pro- 
duced, with  that  which  is  produced  by  the  action  of  pepsin  upon 
the  concentrated  (=6  times)  products  at  36  degrees  was  shown 
by  the  following  experiments. 

Thirty  cubic  centimeters  of  15  per  cent  pepsin  (Gruebler's 
puriss.  sice.)  were  added  to  150  cc.  of  the  unconcentrated  prod- 
ucts of  the  complete  peptic  hydrolysis  of  JV/50  sodium  caseinate 
and  the  mixture  was  kept  at  65  degrees  for  48  hours  in  the 
presence  of  excess  of  toluol.  The  resulting  precipitate  was  col- 
lected on  a  hardened  filter  paper  and  washed  with  distilled  water 
until  the  washings  were  colorless;  it  was  then  dissolved  by  allow- 
ing dilute  sodium  hydrate  to  pass  through  the  filter  and  repre- 
cipitated  by  allowing  this  filtrate  to  pass  into  a  beaker  contain- 
ing excess  of  dilute  acetic  acid.  The  precipitate  thus  obtained 
was  collected  on  a  hardened  filter  paper,  washed  with  large 
volumes  of  water,  alcohol,  and  ether  and  dried  over  CaCk  and 
later  over  H2S04.  The  product  was  a  greyish-white,  friable 
hygroscopic  powder  resembling  in  its  physical  properties  and 
precipitation-reactions  "paranuclein  A"  It  was  analysed  for 
phosphorus  by  Neumann's  method  and  found  to  contain  1.65 


434  CHEMICAL  DYNAMICS 

per  cent  of  PzO?>,  thus  agreeing  closely  with  paranuclein  A  and 
with  the  substance  synthesized  at  36  degrees  from  the  concen- 
trated products  of  the  hydrolysis  of  paranuclein. 

Furthermore,  the  change  in  the  refractive  index  of  AT/50  KOH 
which  is  brought  about  by  the  introduction  of  one  per  cent  of 
this  synthetic  substance  is  identical  with  that  which  is  brought 
about  by  the  introduction  of  one  per  cent  of  paranuclein,  para- 
nuclein A  or  of  the  substance  synthesized  at  36  degrees,  namely 
0.00140  for  sodium  light  (54). 

A  reversion  of  hydrolysis  can  be  brought  about,  therefore, 
even  in  the  diluted  products  of  the  complete  peptic  hydrolysis 
of  N/10  solutions  of  the  alkali  caseinates  (diluted,  since  varying 
amounts  of  pepsin  solutions  were  added  to  the  solution  of  products) 
by  the  addition  of  0.5  cc.  of  15  per  cent  pepsin  to  10  cc.  of 
products  (final  concentration  of  pepsin  0.75  per  cent)  and  keeping 
the  mixture  for  24  hours  at  65  degrees,  while  it  requires  15  cc. 
of  10  per  cent  pepsin  in  100  cc.  of  mixture  (final  concentration 
of  pepsin  1.5  per  cent)  to  bring  about,  in  24  hours,  reversion  of 
the  hydrolysis  at  36  degrees  in  a  solution  of  products  which  has 
been  concentrated  four  or  five  times. 

It  is  an  extremely  significant  fact  that  the  synthesis  which 
occurs  at  65  degrees  does  so  at  a  temperature  from  10  to  15  de- 
grees in  excess  of  that  at  which,  according  to  the  majority  of 
authors,  pepsin  is  rapidly  and  completely  deprived  of  its  proteo- 
lytic  activity.*  True,  the  destruction  even,  at  this  temperature, 
must  be  a  matter  of  time,  and  one  might  be  inclined  to  believe 
that  a  short  period  of  very  intense  action  at  65  degrees  produced, 
in  the  above  experiments,  a  similar  result  to  the  much  more  pro- 
longed but  weaker  action  of  pepsin  at  36  degrees.  The  facts  are 
not  in  favor  of  this  view,  however,  since  the  appearance  of  the  pre- 
cipitate which  marks  a  certain  stage  in  the  reversion  does  not  occur 
(if  the  pepsin  is  not  too  concentrated)  until  the  solution  has  been 
standing  at  65  degrees  for  two  or  three  hours,  and  it  progressively 
increases  in  amount  for  over  24  hours.  It  appears  that  the  active 
agent  in  reversion  is  not  identical  with  the  active  agent  in  hydrolysis. 

*  Cf.  Oppenheimer  (45).-  From  the  determinations  of  Schwarz  (64)  it 
appears  that  concentrated  (10  per  cent)  solutions  of  pepsin  are  deprived  of 
their  power  of  accelerating  protein  hydrolysis  and  at  the  same  time  acquire 
considerable  power  of  inhibiting  the  activity  of  unaltered  pepsin  after  having 
been  heated  to  60  degrees  for  5  minutes. 


REVERSION  OF  HYDROLYSIS 


435 


Additional  experiments  were  undertaken  by  myself  and  Biddle 
(55)  with  a  view  to  further  elucidating  the  relationship  between 
the  paranucleins  and  the  synthetic  substances  which  I  have 
described.  We  determined  their  carbon,  hydrogen  and  nitrogen 
content,  with  the  results  tabulated  below*;  for  the  purpose  of 
comparison  analyses  of  paranuclein  by  Lubavin  (41)  are  also 
included  in  the  table. 


Paranuclein 
(Lubavin), 
per  cent 

Paranuclein 
(Robertson  and 
Biddle), 
per  cent 

Paranuclein  A 
from  paranu- 
clein, 
per  cent 

Substance 
synthesized 
36  degrees, 
per  cent* 

Substance 
synthesized 
70  degrees, 
per  cent 

c 

48.5 

49.98 

49.47 

53.39 

49.99 

H 

7.1 

7.20 

6.80 

7.80 

7.00 

N 

13.3 

12.80 

12.50 

13.00 

13.10 

*  Three  separate  preparations  of  this  substance  were  made,  the  figures  given  (except  that  for  N 
which  was  only  determined  in  the  first  preparation)  are  those  obtained  with  the  preparation  which 
had  been  subjected  to  the  most  rigorous  purification. 

It  is  evident  that  the  carbon,  hydrogen,  and  nitrogen-contents 
of  paranuclein,  paranuclein  A  and  the  substance  synthesized 
at  70  degrees,  are  so  closely  similar  that  they  may  be  considered 
identical;  they  also  agree  well  with  the  original  analyses  of 
Lubavin.  The  product  obtained  at  36  degrees,  however,  con- 
tains a  very  appreciably  higher  percentage  of  carbon.  Since  the 
carbon-content  of  this  preparation  was  found  to  sink  somewhat 
during  successive  purifications  it  appears  possible  that  the  high 
carbon  is  due  to  contamination  with  some  impurity,  possibly  a 
coagulose  (Cf.  below).  The  product  which  is  formed  at  36 
degrees  is  deposited  from  the  mixture  more  slowly  than  that 
which  is  formed  at  70  degrees,  hence  it  would  be  more  liable  to 
carry  down  contaminations. 

The  objection  has  been  urged  by  Bayliss  (5)  and  by  Rohonyi 
(57)  that  it  is  impossible  to  adequately  identify  a  protein  by 
purely  analytical  similarities.  They  point  out  that  Gruebler's 
pepsin  contains  a  substance  which  is  coagulable  by  heating  and 
that  after  this  substance  has  been  removed  the  pepsin  solution 
no  longer  retains  the  power  of  causing  the  appearance  of  a  pre- 
cipitate when  mixed  with  a  solution  of  the  products  of  the  peptic 
hydrolysis  of  casein.  Merck's  pepsin,  on  the  other  hand,  yields 

*  For  quantitive  details  of  the  methods  employed  in  preparing  these  sub- 
stances in  bulk  consult  the  communication  just  cited. 


436  CHEMICAL  DYNAMICS 

no  coagulum  on  heating  and  yields  no  precipitate  upon  admixture 
with  a  concentrated  solution  of  the  products  of  the  peptic 
hydrolysis  of  casein.  From  these  facts  they  infer  that  the  sub- 
stance which  I  believed  to  have  been  enzymatically  synthesized 
paranuclein  was,  in  reality,  a  compound  of  the  heat-coagulable  pro- 
tein contained  in  Gruebler's  pepsin  with  the  caseoses  contained  in 
the  solution  of  the  products  of  the  peptic  hydrolysis  of  casein. 

While  the  analytical  characterization  and  identification  of  a 
protein  substance  is  extremely  difficult  and  inconclusive,  the 
phenomena  of  specific  immunization,  on  the  other  hand,  afford 
us  a  method  of  identifying  protein  substances  which,  so  far  as 
our  knowledge  at  present  extends,  is  decisive  and  extremely 
sensitive.  The  investigations  of  Wells  (75)  and  of  Wells  and 
Osborne  (76)  have  especially  demonstrated  the  high  degree  of 
specificity  displayed  by  the  immune-bodies  which  appear  in  the 
circulation  of  animals  as  a  result  of  repeated  administration  of 
foreign  proteins.  It  would  appear  to  be  thoroughly  established 
that  only  protein  substances  are  antigenic,  that  protein  split- 
products  below  a  certain  degree  of  complexity  are  non-antigenic, 
and  that  the  immune-bodies  which  are  developed  in  response 
to  immunization  against  a  given  protein  will  react  only  with 
that  protein  or  with  a  protein  which  contains,  as  an  integral 
portion  of  its  molecule,  a  large  fraction  of  the  molecule  of  the 
protein  employed  in  immunization. 

Advantage  was  taken  of  these  facts  by  Gay  and  Robertson 
(20)  to  investigate  more  fully  the  question  of  the  identity  or 
non-identity  of  the  enzymatically  synthesized  paranuclein  with 
the  paranuclein  which  results  from  the  partial  hydrolysis  of 
casein.  They  found,  employing  both  anaphylaxis  and  alexin- 
fixation  as  indicators  of  the  development  of  immune-bodies,  that 
the  products  of  the  complete  peptic  hydrolysis  of  casein  are 
toxic  for  normal  animals.  They  have,  however,  no  antigenic 
property,  nor  any  specific  intoxicating  effect  upon  animals  sen- 
sitized to  themselves  or  to  paranuclein.  Gruebler's  pepsin  itself 
is  likewise  non-antigenic.  Paranuclein  and  the  enzymatically 
synthesized  paranuclein,  however,  both  yield  specific  antibodies 
and  these  antibodies  react-  interchangeably  with  either  substance. 
These  results  would  appear  to  yield  unequivocal  evidence  of  the 
occurrence  of  a  genuine  protein  synthesis  under  the  conditions 
outlined  above. 


COAGULOSES  AND  PLASTEINS  437 

The  question  remains  as  to  why  this  synthesis  is  not  achieved, 
or,  at  least,  does  not  result  in  the  production  of  a  visible  pre- 
cipitate, when  Merck's  pepsin  is  substituted  for  Gruebler's  prep- 
aration. While  a  positive  answer  to  this  question  cannot  as  yet 
be  advanced  with  confidence,  it  would  appear  to  offer  no  very 
valid  reason,  especially  in  view  of  the  experiments  of  Gay  and 
Robertson,  for  deciding  that  enzymatic  synthesis  does  occur 
under  defined  conditions  in  mixtures  of  Gruebler's  pepsin  and 
solutions  of  the  products  of  the  complete  peptic  hydrolysis  of 
casein.  It  would  appear,  on  the  contrary,  to  afford  additional 
justification  for  the  view,  expressed  above,  that  the  active  agent 
in  the  reversion  is  not  identical  with  the  active  agent  in  hydrolysis. 
The  former  substance  is  present  in  Gruebler's  pepsin,  but  is 
absent  from  Merck's  preparation. 

The  occurrence  of  synthetic  processes  (i.e.,  the  reverse  of 
hydrolyses)  in  mixtures  of  pepsin  and  concentrated  solutions  of 
the  peptic  split-products  of  proteins  has  also  been  demonstrated, 
in  quite  a  different  manner,  by  Henriques  and  Gjaldbaek  (27). 
These  observers  have  shown  that  if  pepsin  be  added  to  an  acid 
and  concentrated  solution  of  the  peptic  split-products  of  a  protein, 
a  synthetic  process  (union  of  COOH  and  H2N  groups)  occurs, 
as  indicated  (a)  by  a  progressive  increase  in  the  substances  pre- 
cipitable  by  tannic  acid,  and  (6)  by  a  decrease  in  the  groups 
(H2N  — )  which  are  capable  of  reacting  with  formaldehyde.  The 
process  occurs  at  all  temperatures  lying  between  5  and  70  degrees. 
The  more  completely  split  the  material  in  the  digest  is  to  begin 
with  the  less  complex  are  the  products  which  result,  but  at  the 
same  time  their  mass  is  greater.  The  most  complex  substances 
which  are  formed  in  consequence  of  this  reversion  do  not  contain 
much  more  formol-titratable  nitrogen  than  the  original  proteins. 
They  have,  moreover,  shown  (28)  that  the  synthesis  is  reversible, 
more  dilution  leading  to  a  progressive  increase  in  the  formol- 
titre.  Furthermore,  a  decrease  in  the  formol-titre  also  occurs 
when  trypsin  is  added  to  a  concentrated  solution  of  the  products 
of  the  peptic  hydrolysis  of  protein. 

2.  The  Probable  Nature  of  the  Coaguloses  and  Plasteins.  — 
It  will  be  observed,  on  referring  to  the  analytical  data  obtained 
by  Robertson  and  Biddle  (55),  that  the  carbon  content  of  the 
paranucleins  (49-50  per  cent)  is  considerably  lower  than  that 
of  the  proteins  (51-55  per  cent).  In  this,  and  in  other  respects, 


438  CHEMICAL  DYNAMICS 

paranuclein  differs  very  fundamentally  from  the  coaguloses,  for 
the  carbon-content  of  the  coaguloses,  so  far  from  being  abnor- 
mally low,  is  abnormally  high  (58-59  per  cent,  Kurajev).  The 
syntheses  described  above  are  therefore  not  merely  examples  of 
"plastein"  or  "coagulose"  formation. 

Kuhne  and  Chittenden  (35)  observed  that  if  the  portion  of  a 
protein  which  is  not  acted  upon  by  prolonged  digestion  with 
pepsin  be  subsequently  subjected  to  tryptic  digestion  a  jelly 
separates  out  which  they  termed  the  "  anti-albumid  coagulum" 
and  which  is  very  slowly  attacked  by  trypsin.  Okunev,  Lavrov, 
Savjalov,  Kurajev  and  Umber  (44)  (38)  (39)  (62)  (63)  (36)  (50) 
have  prepared  similar  substances  by  acting  upon  peptic  digests 
with  rennet,  or  with  salts  (ammonium  sulphate,  Cf.  Umber, 
loc.  cit.),  or  with  finely  divided  insoluble  powders  (lycopodium), 
and  have  termed  them  "plasteins"  or  "  coaguloses."  These  sub- 
stances are  albumoses  and  not  proteins,  .since  they  are  digested 
by  erepsin  (Lavrov  and  Salaskin).  Savjalov  finds  that  the 
amount  of  plastein  which  is  formed  runs  parallel  with  the  quantity 
of  hetero-albumose  which  the  protein  yields,  that  is,  to  the 
quantity  of  the  ^difficultly-digestible  anti-fraction. 

All  the  circumstances  of  plastein  formation  point  toward  a 
modified  hydrolysis  as  the  condition  of  their  formation.  No 
preliminary  concentration  of  the  system  is  requisite,  and  no 
rise  in  temperature.  It  appears  probable,  therefore,  that  plas- 
tein formation  is  not  a  reversion  of  protein  hydrolysis,  as  some 
authors  have  suggested,  but  rather  a  coagulation  due  to  partial 
hydrolysis  induced  by  the  rennet-like  action  of  ferments  upon 
certain  albumoses  or  peptids,*  these  playing  the  part  which  is 
played,  in  our  more  familiar  experience,  by  calcium  caseinate. 

In  satisfactory  harmony  with  this  view  Herrmann  and  Chain 
(30)  have  found  that  the  antisera  from  rabbits  immunized  with 
plastein  derived  from  Witte's  peptone,  tested  by  the  precipitin 
reaction,  react  with  plastein,  not  only  from  Witte  peptone  but 
from  other  proteins,  for  example,  edestin,  serum  albumin,  egg- 
albumin  and  the  globulin  from  almonds.  They  do  not  react, 
however,  with  Witte  peptone  itself  nor  with  casein.  In  this 
respect  the  plasteins  differ  very  decisively  from  enzymatically 
synthesized  paranuclein,  for  the  antiserum  to  synthetic  para- 
nuclein reacts  not  only  with  normal  paranuclein  but  also  with 
*  Regarding  the  probable  mode  of  action  of  rennet  (Cf .  previous  chapter,  6). 


CHEMICAL  MECHANICS  OF  SYNTHESIS  439 

the  protein,  casein,  from  which  paranuclein  is  derived  and  of 
which  it  forms  a  large  and  integral  proportion.  The  plasteins, 
on  the  other  hand,  would  appear  to  form,  antigenically  speaking, 
a  group  of  substances  which  are  very  closely  related  to  one 
another  and  probably  contain  a  large  common  fraction. 

3.  The  Chemical  Mechanics  of  the  Fermentative  Synthesis 
of  Proteins.  —  We  have  seen  in  section  1  that  in  instances  of 
"typical"  catalytic  hydrolysis,  although  the  degree  of  hydrolysis 
in  dilute  systems,  under  the  influence  of  a  catalysor,  may  be  prac- 
tically complete,  yet,  if  the  solution  of  the  products  be  concen- 
trated, the  station  of  equilibrium  will  be  shifted  and  may  be 
shifted  to  such  an  extent  that  the  pure  products  are  not  in 
equilibrium  and  an  appreciable  quantity  of  substrate  may  be  re- 
stored to  the  system  under  the  influence  of  a  catalysor.  Hence 
it  was  natural  to  suppose,  as  Taylor  did  (loc.  cit.)  that  the  synthe- 
sis of  protamin,  accomplished  through  the  action  of  trypsin  upon 
the  concentrated  products  of  its  hydrolysis,  is  an  example  of  the 
reversion  of  a  " typical"  catalysed  reaction  in  which  the  catalysor 
plays  no  part  in  determining  the  final  equilibrium.  Neverthe- 
less it  must  be  recollected  that  the  validity  of  this  view  can  by 
no  means  be  regarded  as  proven  until  it  has  been  shown  that  the 
station  of  equilibrium  in  the  presence  of  the  catalysor  is  definitely 
the  same  as  it  is  in  its  absence,  and  this  has  not  been  done; 
indeed,  the  great  technical  difficulty  of  the  problem,  as  it  at 
present  appears  to  us,  discourages  the  attempt.  Moreover, 
there  are  many  facts,  a  number  of  which  have  been  alluded  to 
in  the  previous  chapter,  which  speak  very  decidedly  against  the 
view  that  the  fermentative  syntheses  of  proteins  are  instances 
of  " typical"  catalytic  reversion.  To  those  which  have  already 
been  dwelt  upon  may  be  added  the  following. 

We  have  seen  that  the  velocity  of  protein  hydrolysis  is,  under 
certain  conditions,  directly  proportional  to  the  concentration  of 
the  ferment,  under  others  proportional  to  the  square  root  of  the 
ferment-concentration  (Chap.  XVI,  section  3).  If  the  proteolytic 
ferments  act  as  " typical"  catalysors  and  do  not  in  any  way  affect 
the  final  equilibrium  in  the  system,  then  the  velocity  of  synthesis 
must  also  vary  directly  or  as  the  square  root  of  the  ferment- 
concentration,  for  otherwise  the  ratio  between  the  velocity  con- 
stants of  the  forward  and  opposed  reactions  would  be  a  function 
of  the  ferment-concentration  and,  since  the  station  of  equilibrium 


440  CHEMICAL  DYNAMICS 

is  determined  by  this  ratio,  the  equilibrium  of  the  system  would 
also  be  a  function  of  the  ferment-concentration.  The  synthesis 
of  paranuclein  A  through  the  agency  of  pepsin  affords  us  an 
opportunity  of  determining  the  influence  of  the  ferment-concen- 
tration upon  the  velocity  of  protein  synthesis,  since  the  product 
is  rapidly  formed  and  can  readily  be  determined  quantitatively. 
The  following  was  the  experimental  procedure: 

The  products  of  the  complete  peptic  hydrolysis  of  N/ 10 
potassium  hydrate  neutralized  to  litmus  by  casein*  were  evapo- 
rated to  one-sixth  of  their  volume  and  filtered.  Seventy-five 
cubic  centimeters  of  the  clear,  deep  yellow  filtrate  were  placed 
in  each  of  six  flasks  and  to  each,  respectively  0,  5,  10,  15,  20  and 
25  cc.  of  10  per  cent  pepsin  (puriss.  sice.  Gruebler's)  were  added, 
and  the  total  volume  in  each  flask  was  made  up  to  100  cc.  by  the 
addition  of  distilled  water.  After  the  addition  of  toluol,  the 
tightly-stoppered  flasks  were  set  aside  at  36  degrees  for  22  hours. 
At  the  end  of  this  time  the  flasks  containing  25  and  20  cc.  of 
10  per  cent  pepsin,  respectively,  contained  heavy  precipitates, 
while  that  containing  15  cc.  contained  a  precipitate  and  those 
containing  5  and  10  cc.  had  undergone  no  change  other  than  a 
slight  increase  in  opacity.  The  contents  of  the  flasks  were  now 
filtered  through  rapid-filtering  papers  and  the  precipitates  were 
washed  with  distilled  water  until  colorless  filtrates  were  obtained; 
in  all  cases  the  filtrates  gave  no  precipitates  or  increase  in  opal- 
escence  upon  the  addition  of  acetic  acid.  The  filters  were  then 
washed  with  10  cc.  of  JV/10  potassium  hydrate  and  the  filtrates 
were  collected  in  water  containing  20  cc.  of  N/W  acetic  acid. 

*  Prepared  as  follows:  To  6  litres  of  JV/50  sodium  or  potassium  caseinate, 
neutral  or  faintly  acid  to  litmus,  were  added  two  gram  of  Gruebler's  pepsin 
puriss.  sice,  which  had  previously  been  dissolved  in  a  little  water.  This 
solution,  after  thorough  mixing,  was  allowed  to  stand  at  36  degrees  for  10 
days,  2  more  grams  of  pepsin  being  added  after  the  first  4  days  (hi  the 
presence  of  toluol),  and  was  then  sterilized  by  steam  at  100  degrees  and 
filtered  through  hardened  filter  paper.  To  the  filtrate  were  then  added  two 
more  grams  of  pepsin,  dissolved,  as  in  the  previous  cases,  in  a  little  water, 
toluol  introduced,  and  the  solution  was  again  allowed  to  stand  at  36  degrees  for 
7  to  8  days;  it  was  then  again  sterilized  by  steam  at  100  degrees  and  filtered 
through  hardened  filter  paper.  The  filtrate  thus  obtained  is  of  a  clear  yellow 
color  with  little  or  no  opalescenca  and  gives  no  trace  of  a  precipitate  or 
opalescence  upon  the  addition  of  acetic  acid  either  before  or  after  neutralization 
with  alkali;  hence  both  casein  and  paranuclein  are  completely  absent  from 
the  solution. 


CHEMICAL  MECHANICS  OF  SYNTHESIS 


441 


The  filter  papers  were  then  thoroughly  macerated  in  dilute 
potassium  hydrate  and  the  magma  thus  prepared  was  filtered 
and  washed  with  distilled  water,  the  filtrate  and  washings  being 
collected  in  the  beaker  which  received  the  first  washings  with 
TV/ 10  potassium  hydrate.  Care  was  taken  to  prevent  more  than 
a  slight  excess  of  acetic  acid  from  being  finally  present  in  this 
beaker.  The  precipitate  which  formed  in  the  beaker  settled  rapidly 
in  small  flocculi,  was  collected  upon  a  soft  rapid-filtering  paper 
and  thoroughly  washed  with  distilled  water  until  the  washings 
were  neutral  to  litmus.  In  all  cases  the  filtrates  and  washings 
were  perfectly  clear  and  free  from  protein.  The  papers  and 
precipitates  thus  obtained  were  macerated  in  water  containing 
a  known  quantity  (10  cc.)  of  TV/10  potassium  hydrate,  the  magma 
thus  obtained  was  diluted  to  about  200  cc.,  phenolphthalein 
(4  drops  of  2  per  cent  alcoholic  solution)  was  added  and  the  solu- 
tions thus  prepared  were  titrated  to  neutrality  with  N/ 10  hydro- 
chloric acid.  A  weighed  amount  (227  milligrams)  of  paranuclein 
A  prepared  in  the  manner  described  in  section  1  was  dissolved  in 
about  100  cc.  of  distilled  water  containing  exactly  10  cc.  of  TV/10 
potassium  hydroxide,  and  the  solution  was  titrated  to  neutrality 
with  TV/ 10  hydrochloric  acid  and  phenolphthalein  indicator;  in 
this  way  it  was  found  that  1  gram  of  paranuclein  A  neutralizes 
4.8  cc.  of  TV/10  potassium  hydroxide*;  hence  1  cc.  of  TV/10  alkali 
=  0.208  gram  of  paranuclein  A  and  we  can  estimate  from  the 
determinations  described  above  the  amount  of  paranuclein  A 
produced  in  each  of  the  mixtures  by  varying  amounts  of  pepsin. 
The  following  were  the  results  obtained: 


Amount  of  pepsin  in  100  cc.  of 
solution 

Milligrams  of  paranuclein  A  pro- 
duced at  the  end  of  22  hours 

25  cc.  of  10  per  cent   .      .  . 

296 

20  cc.  of  10  per  cent  

210 

15  cc.  of  10  per  cent  

162 

10  cc.  of  10  per  cent  

0 

5  cc.  of  10  per  cent  

0 

0  cc.  of  10  per  cent  

0 

*  I  have  found  that  upon  standing  in  the  presence  of  excess  of  alkali,  the 
amount  of  alkali  neutralized  by  a  given  quantity  of  paranuclein  A  increases 
slightly.  I  have  been  unable  to  determine  whether  this  phenomenon  is  due  to 
the  slow  dissolving  of  microscopic  suspended  particles  or  whether  it  is  due  to 


442  CHEMICAL  DYNAMICS 

From  these  results  it  is  clear  that  the  velocity  of  reversion  is 
by  no  means  directly  proportional  to  the  concentration  of  pepsin, 
nor  even  proportional  to  the  square  root  of  the  ferment-concen- 
tration. While  the  velocity  of  synthesis  in  the  most  concentrated 
solutions  (25  to  15  cc.  of  10  per  cent  pepsin  in  100  cc.)  roughly 
approximates  to  direct  proportionality  to  the  concentration  of 
ferment,  in  the  more  dilute  solutions  (10  to  5  cc.  of  10  per  cent 
pepsin  in  100  cc.)  the  velocity  of  synthesis  falls  off  with  extraor- 
dinary rapidity  as  the  concentration  of  the  ferment  diminishes. 
Making  every  possible  allowance  for  experimental  error  arising 
out  of  loss  of  material  during  the  estimation,  an  increase  in 
pepsin-concentration  from  1.0  per  cent  to  1.5  per  cent  multiplies 
the  velocity  of  the  synthesis  over  10  times,  while  an  increase  in 
pepsin  concentration  from  1.5  per  cent  to  2.5  per  cent  only 
doubles  it;  these  facts  are  obviously  irreconcilable  alike  with 
direct  proportionality  between  the  velocity  of  synthesis  and  the 
concentration  of  ferment,  and  with  the  Schiitz  rule  of  propor- 
tionality to  the  square  root  of  the  concentration  of  the  ferment. 
The  velocity  of  reversion  does  not  bear  the  same  relation  to  the 
concentration  of  ferment  that  the  velocity  of  hydrolysis  does; 
hence  the  ratio  of  the  velocity  constants  of  hydrolysis  and  re- 
version must  be  dependent  upon  the  concentration  of  the  ferment, 
or,  in  other  words,  the  equilibrium  between  protein  and  its  products 
must,  to  some  extent,  be  altered  by  pepsin. 

We  have  seen  that  a  reversion  of  the  hydrolysis  of  paranuclein 
can  be  brought  about  without  any  previous  concentration  of  the 
products  of  its  hydrolysis,  provided  the  temperature  be  raised 
to  60-70  degrees.  In  the  light  of  this  fact  there  can  be  little 
doubt  that  a  shift  in  the  station  of  equilibrium  between  para- 
nuclein A  and  its  products  occurs  as  a  result  of  the  addition  of 
pepsin  and  that  this  shift  in  equilibrium  is  favored  by  a  rise  in 
temperature. 

That  a  shift  in  equilibrium  is  involved  even  at  lower  tempera- 
hydrolysis  (56).  The  number  of  cubic  centimeters  neutralized  by  one  gram 
given  above  is  the  lower  figure,  obtained  directly  after  complete  solution, 
judged  by  the  disappearance  of  obvious  particles  within  the  solution.  I  have 
obtained  figures  as  high  as  5.2-  after  allowing  the  solution  to  stand  for  about 
an  hour  at  a  warm  temperature  before  titration.  The  titrations  in  the  experi- 
ment described  were  performed  immediately  after  the  complete  disappearance 
of  obvious  particles. 


CHEMICAL  MECHANICS  OF  SYNTHESIS  443 

tures  is  shown  by  the  fact  that  if  a  sufficiency  of  dry  pepsin*  be 
added  to  the  unconcentrated  products  of  the  complete  peptic 
hydrolysis  of  N/W  sodium  caseinate  synthesis  will  occur  at  36 
degrees.  I  have  found  about  2  grams  of  pepsin  (Gruebler's 
puriss.  sice.)  per  hundred  cubic  centimeters  sufficient  for  this 
purpose.  That  the  equilibrium  is  still  further  shifted  by  a  rise 
in  temperature  is  shown  by  the  following  experiment : 

To  300  cc.  of  the  unconcentrated  products  of  the  peptic  hy- 
drolysis of  N/ 10  sodium  caseinate  were  added  6  grams  of  dry 
pepsin.  After  48  hours  at  36  degrees  (in  the  presence  of  excess 
of  toluol)  a  precipitate  had  formed,  while  the  supernatant  fluid 
remained  somewhat  opalescent;  after  6  days  the  supernatant 
fluid  was  quite  clear  and  a  bulky  precipitate  lay  at  the  bottom 
of  the  flask;  the  clear  fluid  was  now  decanted  from  the  precipi- 
tate and  divided  into  two  parts;  the  one  was  kept  at  36  degrees 
and  the  other  at  65  degrees;  within  8  hours  a  precipitate  had 
formed  in  the  latter,  the  supernatant  fluid  being  strongly  opal- 
escent, while  the  part  of  the  solution  which  remained  at  36 
degrees  had  developed  no  trace  of  precipitate  or  opalescence 
after  a  period  of  two  weeks.  It  is  clear,  therefore,  that  the 
system  had  arrived  at  equilibrium  at  36  degrees  before  the  fluid 
was  decanted,  since  this  fluid  must  have  been  " saturated"  with 
paranuclein  A  (soluble  in  these  acid  solutions  only  to  an  unde- 
tectable  extent)  and  any  further  formation  of  paranuclein  A 
would  have  resulted  in  an  increase  in  opalescence  if  not  in  actual 
precipitation.  This  did  not  occur,  however,  even  during  a  period 
of  two  weeks.  Yet  at  65  degrees  a  fairly  abundant  precipitate 
was  produced  within  8  hours. 

It  is  clear,  therefore,  that  in  the  enzymatic  synthesis  of  para- 
nuclein A  a  marked  shift  in  equilibrium  is  involved,  and  we 
must  therefore  definitely  abandon  the  view  that  pepsin  is  a 
" typical"  catalysor  which  does  not,  to  an  appreciable  extent, 
participate  in  the  reactions  which  it  catalyses. 

The  existence  of  a  similar  influence  of  trypsin  upon  the  equi- 
librium in  protein  solutions  is  possibly  indicated  by  the  investi- 
gations of  Walters  (73),  who  has  shown  that  whereas  the  time- 
relations  of  the  hydrolysis  of  casein*  by  trypsin  are  very  accurately 

*  Dry,  that  is  to  say,  not  in  solution,  for  otherwise  water  is  added  to  the 
system  and  the  solution  of  products  is  actually  diluted  by  the  addition  of  the 
ferment. 


444  CHEMICAL  DYNAMICS 

defined  by  the  mono-molecular  formula,  terminating  only  when 
hydrolysis  is  practically  complete,  the  autohydrolysis,  on  the 
contrary,  which  caseinates  undergo  in  aqueous  solution  in  the 
absence  of  enzymes  falls  off  in  velocity  with  time  much  more 
rapidly  than  would  be  indicated  by  the  monomolecular  formula, 
so  that  the  process  comes  to  a  stop  when  a  very  large  proportion 
of  the  protein  is  still  unhydrolysed.  The  products  of  the  tryptic 
hydrolysis  of  casein  exert,  moreover,  an  almost  negligible  effect 
upon  the  velocity  of  autohydrolysis,  even  when  added  in  very 
high  concentration.  We  must  infer,  therefore,  either  that  trypsin, 
in  dilute  solutions,  shifts  the  equilibrium  between  protein  <=^ 
products  towards  the  right,  or  else  that  the  rapid  slowing-down 
of  the  autohydrolysis  is  due  to  the  attainment  of  a  "  false  equi- 
librium," i.e.,  an  indefinite  delay  in  the  attainment  of  equi- 
librium due  to  the  interposition  of  a  specifically  slow  reaction 
in  a  series  of  catenary  reactions,  each  one  of  which  utilizes  the 
products  of  the  preceding  reaction. 

4.  Reciprocal  Catalysis.  —  We  have  seen  that  enzymatic 
synthesis  of  paranuclein  A  may  occur  at  temperatures  10  to  15 
degrees  in  excess  of  that  at  which  the  hydrolysing  activity  of 
pepsin  is  rapidly  and  completely  destroyed.  This  invites  adop- 
tion of  the  view,  previously  expressed  by  Euler  (17)  (18)  that 
the  synthesizing  and  hydrolysing  forms  of  the  enzyme  are  not 
identical.  Euler  believes  that  two  varieties  of  every  hydrolysing 
enzyme  exist,  the  one  accelerating,  primarily,  hydrolysis,  the  other 
accelerating,  primarily,  synthesis,  and  that  under  definite  condi- 
tions a  definite  equilibrium  exists  between  the  two  enzymes.  He 
bases  this  view,  in  the  main,  upon  the  non-constancy  of  the 
velocity-constant  of  certain  enzyme-accelerated  reactions  with 
varying  substrate-concentration,  and  upon  the  non-identity  of 
the  synthesized  product  (isomaltose)  derived  by  the  action  of 
maltase  upon  concentrated  glucose  with  the  maltose  from  which 
the  glucose  was  derived  by  hydrolysis,  and  of  the  product  (iso- 
lactose)  obtained  by  the  action  of  lactase  upon  a  concentrated 
solution  of  galactose  and  glucose  with  the  lactose  from  which 
the  galactose  and  glucose  are  derived.*  Euler  believes  that  the 
"  anti-trypsins "  found  in -serum  and  in  egg-white  are  simply  the 
synthesizing  form  of  the  enzyme  and  that  the  anti-pepsin  and 

*  It  is  stated,  however,  that  even  sulphuric  acid,  acting  upon  a  concentrated 
solution  of  glucose,  gives  rise,  not  to  maltose  but  to  isomaltose  (19). 


RECIPROCAL  CATALYSIS  445 

anti-trypsin  which  appear  in  the  circulation  after  the  injection 
of  the  enzymes  do  so  as  a  result  of  the  production  of  the  synthe- 
sizing form  in  restoration  of  the  equilibrium  between  the  synthe- 
sizing enzyme  and  the  hydrolysing  form. 

The  hypothesis  which  I  have  put  forward  (53)  in  explanation 
of  the  phenomena  of  protein  hydrolysis  and  synthesis  is  a  modi- 
fication and  extension  of  Euler's  hypothesis,  and  involves  the 
view  that  these  processes  are  examples  of  which  I  have  termed 
" reciprocal  catalysis."  The  essential  features  of  this  hypoth- 
esis have  already  been  embodied  in  equations  (A)  to  (D)  in  Chap. 
XV,  section  1.  For  convenience  of  reference  they  may  be  recap- 
itulated here. 

We  know  that  during  or  preceding  the  hydrolysis  of  proteins 
by  proteolytic  enzymes  the  ferment  combines  with  the  substrate 
(previous  chapter,  2).  Furthermore,  the  reaction  consists  in  the 
introduction  of  the  elements  of  water  into  —  COH.N—  bonds 
of  the  protein  molecule,  each  bond  which  is  attacked  yielding 
two  molecules.  We  may  therefore  represent  the  reaction  of 
hydrolysis  as  follows: 

HF 
I 

(A)  -  COH.N  -  +  HFFOH  -»  -  COH.N  - 

I 
FOH 

HF 

I 

(B)  -COH.N »  -COOH  +  H2N-  +  FF   . 

I 
FOH 

while,  subsequently,  the  dehydrated  form  of  the  enzyme  (FF) 
reacts  with  water  to  regenerate  the  hydrated  form : 

(D)  FF  +  H20  +±  HFFOH 

enzymatic  synthesis  of  a  protein  consisting  in  the  reverse  of 
these  reactions. 

The  net  result  of  the  first  two  reactions  is  the  transference  of  the 
elements  of  water  from  the  ferment  to  the  substrate  molecule, 
while,  in  the  third  reaction,  the  ferment  recoups  itself  from  the 
medium.  Provided  the  station  of  equilibrium  in  the  reaction 
(D)  lay  far  enough  to  the  right  and  the  velocity  of  this  reaction 


446  CHEMICAL  DYNAMICS 

measured  from  left  to  right  were  great  compared  with  that  of 
either  of  the  reactions  (A)  and  (B)  measured  from  right  to  left, 
and  provided,  also,  the  quantity  of  enzyme  combined  at  any 
instant  with  the  substrate  were  unappreciable  we  would  obtain, 
for  the  kinetics  of  the  reaction  of  hydrolysis,  the  monomolecular 
equation,  in  which  the  velocity  constant  of  hydrolysis  would 
be  proportional  to  the  ferment-mass.  If  these  conditions  were 
not  fulfilled  we  would  obtain,  provided  the  proportion  of  the 
dehydrated  to  the  hydrated  enzyme  did  not  alter  very  appre- 
ciably during  the  reaction,  the  differential  equation 

dx  kiF  (A  -  x) 


dt       l  +  a(A-x)-ftx2' 
or  its  integrated  form: 

A       ,    a  +  0A_  ft 


(i) 


4         ,.., 

(Cf.  Chap.  XVI.)     For  the  reversion  we  would  obviously  obtain 
the  differential  equation: 

_  dx  = k2Fx* ..... 

dt  ~  l-a(A-x)+ftx2' 

From  equations  (A)  to  (D),  applying  the  mass-law,  it  will  be 
obvious  that  the  quantity  of  enzyme  dehydrated  by  unit  mass 
of  substrate  will  be  dependent  upon  the  mass  of  the  hydrated  form 
which  is  present,  consequently,  the  constant  a  (and  similarly  the 
constant  ft)  will  be  dependent  upon  the  mass  of  ferment  present, 
and  will  also,  if  an  appreciable  shift  in  equilibrium  represented  by 
equation  (D)  occurs  as  a  result  of  the  hydrolysis,  alter  somewhat 
in  value  as  hydrolysis  proceeds  *;  hence  the  final  position  of  equi- 
librium, when  the  velocity  of  reversion  is  equal  to  the  velocity  of 
hydrolysis,  must  be  dependent  upon  the  mass  of  ferment. 

Quite  independently  of  the  mathematical  formulation  of  the 
tune-relations  involved  in  this  process,  it  will  be  clearly  seen  that 
the  presence  of  the  enzyme  must  result  in  a  greater  or  smaller 
shifting  of  the  point  of  equilibrium  between  the  protein  and  its 
products  when  we  reflect  that  the  hydrated  form  HFFOH,  accord- 
ing to  the  above  scheme.,  only  accelerates  the  hydrolysis,  while 
the  anhydrous  form  FF  only  accelerates  the  synthesis,  and  since 

*  It  is  for  this  reason  that  we  cannot  employ  equations  (i)  and  (iii)  to  deter- 
mine the  station  of  equilibrium  in  these  systems. 


RECIPROCAL  CATALYSIS  447 

these  are,  in  general,  present  in  unequal  concentrations,  the  equi- 
librium between  the  forward  and  reverse  reactions  of  protein 
hydrolysis  must  be  shifted.  Since,  however,  for  every  shift  in 
equilibrium,  there  must  be  a  corresponding  expenditure  of  energy, 
the  equilibrium  between  the  anhydrous  and  hydrated  forms  of  the 
enzyme  must  also  be  shifted  by  the  protein;  just  as  the  enzyme 
accelerates  the  hydrolysis  of  the  protein  more  than  its  synthesis 
when  the  hydrated  form  of  the  enzyme  is  initially  present  in  con- 
siderable excess  of  its  anhydrous  form,  so  the  protein  accelerates 
the  dehydration  of  the  enzyme  more  than  its  hydration  because  it 
is  initially  present  in  great  excess  of  its  products  of  hydrolysis. 
This  latter  fact  will  itself  lead  to  a  slowing  of  the  hydrolysis  of  the 
protein  since  the  hydrated  (and  hydrolysis  accelerating)  form  of 
the  enzyme  is  thereby  diminished  in  concentration;  as  the  hydroly- 
sis proceeds,  however,  this  effect  will  diminish;  the  products  of  the 
protein  hydrolysis  will  tend  to  increase  the  proportion  of  the 
hydrated  form  of  the  enzyme  to  the  anhydrous  form,  and  the  rate 
of  hydrolysis  will  increase  at  the  expense  of  the  rate  of  synthesis. 
Ultimately,  it  is  evident  that  a  condition  of  equilibrium  must  be 
reached  in  which  the  station  of  equilibrium  between  the  protein 
and  its  products  is  shifted  further  in  the  direction  protein  — > 
products  than  its  position  in  the  absence  of  the  enzyme,  while  the 
station  of  equilibrium  between  the  hydrated  and  anhydrous  forms 
of  the  enzyme  is  shifted  further  in  the  direction  hydrated  form  — » 
anhydrous  form  than  its  position  in  the  absence  of  the  protein. 
This  position  of  equilibrium  will  depend,  obviously,  upon  the  total 
concentration  of  the  enzyme  and  of  the  substrate,  respectively, 
and  once  attained,  a  further  addition  of  substrate  would  reinaugu- 
rate  the  hydrolysis  of  protein,  it  is  true,  because  the  active  mass  of 
hydrolysable  protein  would  thus  be  increased,  but  it  would  shift 
the  point  of  equilibrium  in  the  direction  products  — >  protein; 
addition  of  enzyme  would  shift  the  station  of  equilibrium  in  the 
direction  protein  — >  products,  as  has  been  found  by  Bayliss  and 
others  (4)  (68).  This  latter  statement,  however,  holds  good  only 
while  the  water  in  the  system  is  in  great  excess  of  the  enzyme,  so 
that  varying  concentration  of  the  enzyme  does  not  appreciably 
affect  the  proportion  subsisting  between  the  hydrated  and  anhy- 
drous forms  at  equilibrium  in  the  absence  of  proteins.  If,  however, 
the  enzyme  be  very  concentrated,  then  the  proportion  of  water  to 
enzyme  (Cf.  Eq.  (D))  will  appreciably  affect  the  equilibrium  be- 


448  CHEMICAL  DYNAMICS 

tween  the  hydrated  and  anhydrous  forms  of  the  enzyme,  and  there 
will  be  present  a  relatively  greater  proportion  of  the  anhydrous 
(synthesis  accelerating)  form  FF;  hence,  under  these  conditions, 
a  further  addition  of  enzyme  will  shift  the  equilibrium  of  the 
protein  in  the  direction  products  — >  protein. 

A  remarkable  feature  of  the  syntheses  of  protein  through 
enzyme  agency  which  have  been  accomplished  by  Taylor  and 
myself  is  the  high  concentration  of  enzyme  which  has  to  be  em- 
ployed; the  reason  for  this  is  now  clear;  the  highly  concentrated 
enzyme  contains  a  greater  proportion  of  the  anhydrous  form  and 
it  shifts  the  equilibrium  of  the  protein  in  the  direction  of  synthesis. 
It  is  now  clear,  also,  why  a  sufficiently  high  concentration  of  en- 
zyme will  actually  bring  about  synthesis  of  protein  in  the  uncon- 
centrated  products  of  the  complete  hydrolysis  of  a  solution  of 
protein,  brought  about  by  the  agency  of  dilute  enzyme.  The 
dependence  of  the  velocity-constant  of  hydrolysis,  calculated  from 
the  monomolecular  formula,  upon  the  initial  concentration  of  the 
substrate  (Cf .  Chap.  XVI,  section  3)  is  also  readily  comprehended, 
since,  as  pointed  out  above,  increase  in  the  concentration  of  sub- 
strate must,  in  the  initial  stages  of  hydrolysis,  lead  to  an  increase 
in  the  proportion  of  the  anhydrous  to  the  hydrated  form  of  the 
enzyme,  and  hence  to  a  diminution  of  the  concentration  of  the 
hydrolysis-accelerating  form  of  the  enzyme;  hence  the  velocity- 
constant  of  hydrolysis  diminishes  with  increasing  substrate- 
concentration. 

6.  The  Influence  of  Temperature  upon  the  Enzymatic  Syn- 
thesis of  Proteins.  —  The  action  of  high  temperatures  in  enhanc- 
ing the  synthesizing  power  of  the  enzyme  while  diminishing  or 
abolishing  its  power  of  accelerating  hydrolysis  may  be  interpreted 
in  either  of  two  ways.  Either  the  high  temperature  destroys  the 
hydrated  form  by  accelerating  its  hydrolysis,  while  leaving  the 
anhydrous  form  unaffected,  so  that  during  the  period  that  the 
anhydrous  is  changing  into  the  hydrated  form  only  the  synthesis 
of  the  protein  is  being  accelerated,  and  not  its  hydrolysis,  or,  more 
probably,  the  high  temperature  actually  shifts  the  equilibrium  of 
reaction  (D)  in  the  direction  HFFOH  — »  FF  +  H2O,  a  conception 
which,  if  we  regard  the  proteolytic  enzymes  as  being  substances 
analogous  to  proteins,  is  in  good  accord  with  our  knowledge  of  the 
influence  of  heat  upon  these  bodies  (Chap.  XII).  The  fact  that 
heating  a  proteolytic  enzyme  leads  to  the  formation  of  substances 


INFLUENCE  OF  TEMPERATURE  449 

which  strongly  retard  protein  hydrolysis  has  been  observed  by  a 
number  of  investigators.  Thus  Schwarz  (64)  has  shown  that  if 
solutions  of  pepsin  be  heated  to  80  degrees  for  some  time  and  then 
added  to  peptic  digests  the  digestion  is  greatly  retarded,  while 
Pollack  (46)  had  previously  obtained,  by  heating  pancreas  extracts 
to  70  degrees,  a  substance  which  greatly  retards  tryptic  hydrolysis 
of  proteins;  he  further  observed  that  this  substance  is  a  colloid 
since  it  does  not  pass  through  the  membrane  of  a  dialysor.  Hensel 
(29)  has  observed  that  if  the  mucous  membrane  of  a  stomach  be 
treated  with  acidulated  water  at  50  degrees,  the  watery  extract 
thus  obtained  contains  an  organic  substance  which  greatly  retards 
peptic  hydrolysis  of  proteins.  Beam  and  Cramer  (6)  have  found 
that  solutions  of  rennet  which  have  been  heated  to  from  56  to 
60  degrees  exert  an  inhibitory  influence  on  the  activity  of  unheated 
rennet.  Bayliss  (4)  has  found  that  heated  trypsin  not  only  greatly 
delays  the  tryptic  hydrolysis  of  casein  but  would  appear  to  in- 
augurate a  chemical  reaction  in  the  reverse  sense,  since  mixtures 
of  heated  trypsin  and  a  casemate,  instead  of  increasing  in  conduc- 
tivity with  time,  at  first  decrease  in  conductivity,  later  slowly 
increasing.  Similar  phenomena  have  been  observed  with  other 
enzymes  (15)  (6). 

The  anti-tryptic  actions  of  such  substances  as  egg-white  (72)  (4), 
normal  blood  serum  (22)  (17)  (49)  (37)  (12)  (24)  and  extracts  of 
intestinal  worms  (13)  and  the  anti-peptic  and  anti-tryptic  actions 
of  the  bodies  produced  in  the  circulation  by  the  injection  of  pepsin 
and  trypsin  into  living  animals  (58)  (1)  (74)  have  usually  been 
attributed  to  the  formation  of  more  or  less  stable  compounds 
between  the  ferment  and  the  anti-ferment.  One  would  be  in- 
clined to  similarly  attribute  the  action  of  heated  solutions  of  pepsin 
and  trypsin  in  inhibiting  these  ferments  to  the  formation  of  com- 
pounds between  the  heated  and  the  normal  ferment  were  it  not 
that  the  heated  ferment,  although  deprived  of  its  power  to  acceler- 
ate hydrolysis  of  protein,  markedly  accelerates  its  synthesis.  The 
inhibitory  action  of  the  heated  ferment  is  thus  clearly  seen  to 
consist  in  bringing  about  a  change  which  is  opposite  in  sense  to 
that  which  is  brought  about  by  the  normal  ferment.*  We  see, 

*  Bayliss,  regarding  the  phenomenon  from  the  standpoint  of  Ehrlich's 
"side-chain"  hypothesis,  has  advanced  the  opinion  that  the  decrease  in  con- 
ductivity which  is  observed  upon  the  addition  of  heated  ferment  to  a  solu- 
tion of  protein  is  to  be  attributed  to  the  destruction  of  the  "zymophore,"  or 


450  CHEMICAL  DYNAMICS 

also,  that  the  loss  of  proteolytic  activity  which  occurs  on  heating 
solutions  of  proteolytic  ferments  is  to  be  attributed,  not  to  auto- 
hydrolysis  of  the  ferment,  but  to  the  partial  or  complete  conversion 
of  the  hydrolysing  enzyme  into  the  synthesizing  form.  From  the 
above  account  of  the  chemical  mechanics  of  the  enzymatic  hydroly- 
sis and  synthesis  of  proteins  it  would  appear  that  the  "inactiva- 
tion"  of  proteolytic  ferments  by  heat  (provided  actual  coagulation 
does  not  occur)  should  be  a  reversible  process,  if  not  in  the  pure 
enzyme-solution,  at  any  rate  in  the  presence  of  products  of  protein 
hydrolysis.*  Owing  to  the  fact  that  the  enzymatic  activity  of 
hydrolysing  enzymes  has  in  the  past  been  identified  with  their 
power  to  accelerate  hydrolysis,  the  application  of  heat  to  enzyme 
solutions  has  always  been  regarded  as  involving  the  "  destruction  " 
of  the  enzymes  and  restoration  of  their  activity  under  varying 
conditions  following  a  return  to  lower  temperatures  has  not  been 
anticipated  and  therefore  has  not  been  looked  for  with  any  especial 
care.  Nevertheless,  Bayliss  (4)  has  observed  that  the  power  of 
heated  trypsin  to  accelerate  protein  hydrolysis  is  to  some  extent 
regained  after  the  heated  enzyme  has  been  mixed  with  protein  for 
some  time,  and  Howell  (33)  has  observed  that  thrombin  is  not 
" destroyed"  by  moderate  heating,  but  is  reversibly  thermolabile 
inasmuch  as  it  regains  its  activity  on  treatment  with  alkali  and 
subsequent  neutralization  at  lower  temperatures.  Gramenisky 
(21)  has  also  observed  that  oxidase  inactivated  by  heating  rapidly 
regains  its  activity  on  standing  at  lower  temperatures.! 

6.  The  Thermodynamics  of  the  Enzymatic  Hydrolysis  and 
Synthesis  of  Proteins.  —  The  thermodynamical  aspect  of  the 
hypothesis  developed  above  is  of  interest.  As  I  have  pointed  out, 
the  shift  in  equilibrium  of  the  system  protein  +±  products  towards 

digesting  group  of  the  ferment  while  the  "haptophore"  group,  by  which  the 
enzyme  attaches  itself  to  the  protein  molecule,  is  unaffected.  The  heated 
ferment  he  terms  "zymoid,"  and  he  believes  that  the  initial  decrease  in  the 
conductivity  of  mixtures  of  casein  and  heated  ferment  is  to  be  attributed  to 
the  formation  of  a  compound  between  the  "zymoid"  and  the  protein.  This 
view,  of  course,  fails  to  account  for  the  reversion  of  protein  hydrolysis  which 
is  brought  about  by  heated  pepsin. 

*  Since,  as  we  have  seen  in  the  preceding  section,  these  will  catalyse  the 
reaction  FF  +  H2O  ->  HFFOH. 

f  In  this  connection  it  is  of  extreme  significance  that  the  oxidase  employed 
by  Gramenisky  behaved,  at  80  degrees,  no'longer  as  an  oxidase  but  as  a  reduc- 
tase,  or  reducing  ferment. 


THERMODYNAMICS  451 

the  right  which  results  from  the  introduction  of  a  proteolytic 
enzyme  into  the  system  must  result  in  a  corresponding  shift  in  the 
equilibrium  of  the  system  anhydrous  enzyme  +±  hydrated  enzyme 
towards  the  left  and  vice  versa.  Since  the  enzyme  is  usually 
present  in  small  concentrations  compared  with  the  protein,  the 
shift  in  the  equilibrium  between  the  two  forms  of  the  enzyme  must 
be  large  compared  with  that  of  the  equilibrium  between  the  protein 
and  its  products;  or  else  the  energy  expended  in  a  shift  of  the  enzyme- 
equilibrium  must  be  great  compared  with  the  energy  expended  in  a 
shift  of  the  protein  equilibrium.  The  latter  appears  to  be  the  more 
probable  view;  since,  otherwise,  the  shift  in  the  protein-equilibrium 
would  probably  be  in  all  cases  too  small  to  be  observed,  and, 
moreover,  we  know  that  the  reaction  of  protein  hydrolysis  is 
only  very  faintly  exothermic  (65)  (23)  (25)  (40)  so  that  the  energy 
change  involved  in  a  shift  in  the  equilibrium  between  protein  and 
the  products  of  its  hydrolysis  is  probably  very  small,  and  a  slight 
shift  in  the  equilibrium  of  a  relatively  very  small  amount  of  enzyme 
might  suffice,  if  the  reaction  of  enzyme  hydration  involved  a 
relatively  considerable  energy-change,  to  provide  the  energy  for  a 
considerable  change  in  the  equilibrium  of  a  relatively  large  mass  of 
protein. 

It  will  be  observed,  therefore,  that  the  processes  of  the  enzymatic 
hydrolysis  and  synthesis  of  proteins  do  not  necessarily  involve 
any  deviation  from  the  laws  of  thermodynamics,  in  spite  of  the 
fact  that  the  enzyme  which  catalyses  these  processes  markedly  in- 
fluences the  equilibrium  between  the  protein  and  its  products.  In 
the  analogous  case  which  is  afforded  by  the  enzymatic  synthesis 
and  hydrolysis  of  fats  Dietz  (14)  assumes  that  because  the  station 
of  equilibrium  is  altered  by  the  presence  of  the  enzyme  the  second 
law  of  thermodynamics  must  be  violated  in  these  systems,  for,  he 
argues,  since  the  catalysor  (enzyme)  can  be  withdrawn  from  the 
system  without  the  expenditure  of  any  work,  it  would  only  be 
necessary  to  allow  the  system  to  come  to  equilibrium  in  the  pres- 
ence of  the  enzyme  and  then  withdraw  the  enzyme  and  allow  it  to 
come  to  its  normal  station  of  equilibrium  and  then  reintroduce  the 
enzyme,  and  so  forth,  to  bring  about  an  endless  cycle  of  changes 
and  to  secure  an  unlimited  quantity  of  energy  without  the  expendi- 
ture of  any  work.  But  in  this  argument,  it  should  be  pointed  out, 
Dietz  assumes  the  answer  to  the  very  question  at  issue;  he  assumes 
in  a  word,  that  the  enzyme  in  question  is  a  "typical"  catalysor, 


452  CHEMICAL  DYNAMICS 

not  entering  to  any  appreciable  extent  into  the  reaction,  and  there- 
fore not  requiring  the  expenditure  of  any  energy  to  withdraw  it 
from  the  system.  Most  investigators  will  prefer  to  assume,  from 
Dietz'  results,  that  lipase  is  not  a  " typical"  catalysor,  but  enters 
appreciably  into  the  reaction  which  it  catalyses  (10)  (11). 

7.  Biological  Applications.  —  It  may  here  be  noted  that  the 
hypothesis  outlined  above  is  not  inconsistent  with  the  generally 
expressed  view  that  the  enzyme  may  be  recovered  without  appreci- 
able loss  of  activity  from  a  protein  digest  which  has  reached  equi- 
librium. Even  if,  in  consequence  of  the  presence  of  protein,  the 
station  of  equilibrium  between  the  two  modifications  of  the 
enzyme  has  been  shifted  in  such  a  direction  as  to  diminish  its 
power  of  accelerating  hydrolysis,  yet  upon  removal  from  the 
system  the  enzyme  would  regain  its  normal  equilibrium,  although, 
of  course,  this  process  might  involve  a  greater  or  a  shorter  period 
of  time.  The  energy  thus  apparently  gained  would  be  slowly  lost 
through  the  slow  (because  of  the  absence  of  catalysors)  regaining 
of  equilibrium  between  the  protein  and  its  products.  The  protein 
+±  products  system  would,  in  other  words,  be  left  " supersaturated" 
in  respect  to  products,  or,  were  the  enzyme  in  the  system  highly 
concentrated,  or  did  it,  for  any  other  reason,  initially  contain  a 
high  proportion  of  anhydrous  (synthesis-accelerating)  form  so  that 
it  gained  power  of  accelerating  hydrolyses  as  a  result  of  the  presence 
of  protein,  then  the  protein  <F±  products  system  would  be  left,  by 
removal  of  the  enzyme,  "  supersaturated "  with  respect  to  protein. 
In  the  former  case  the  anhydrous  <=*  hydrated  enzyme  system 
would  be  left  supersaturated  with  respect  to  the  anhydrous 
(synthesis-accelerating)  form,  in  the  latter,  with  respect  to  the 
hydrated  (hydrolysis-accelerating)  form.  By  mechanical  separa- 
tion of  the  enzyme  and  substrate  at  various  periods  in  living 
tissues  it  is  obvious  that  a  cycle  of  such  operations  could  occur, 
the  enzyme  being  utilized  over  and  over  again,  now  for  hydrolysis 
and  now  for  synthesis.  It  is  obvious  that  if  such  reciprocal  rela- 
tions as  that  outlined  above  find  a  place  in  living  material,  the 
organism  may  be  enabled  thereby  to  temporarily  and  locally  store 
up  large  quantities  of  energy.  The  significance  of  this  possibility 
in  the  general  interpretation  of  life-phenomena  is  patent. 

Finally,  it  may  be  pointed  out  that  the  conceptions  which  I  have 
developed  above  enable  us  to  understand  how  the  living  organism, 
through  temporary  mechanical  or  chemical  separation  and  localiza- 


LITERATURE  CITED  453 

tion  of  the  synthesizing  forms  of  these  enzymes  can  bring  about 
protein-synthesis  even  in  the  presence  of  the  considerable  quantity 
of  water  which  living  tissues  contain.  If  we  regard  the  enzymes 
as  " typical7'  catalysors  then,  as  Bradley  has  pointed  out  (9), 
enzymatic  synthesis  of  fat  (and  the  same  may  be  said  of  the  enzy- 
matic synthesis  of  protein)  could  only  occur  under  conditions 
approximating  to  dessication,  conditions  which  it  is  very  difficult 
to  picture  as  occurring  in  living  tissues. 

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APPENDIX 

THE  TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS 
IN  PROTEIN   SYSTEMS 

The  carrying  out  of  accurate  electrochemical  measurements  in 
solutions  which  contain  proteins  is  attended  by  a  number  of  diffi- 
culties which  result  in  part  from  the  physical  characteristics  of 
such  solutions  and  in  part  from  the  instability  of  the  proteins  and 
their  tendency  to  undergo  hydrolysis.  The  following  is  a  descrip- 
tion of  the  technique  which  I  have  found  it  best  to  employ  when 
obtaining  such  measurements  in  solutions  of  casein  especially*  but 
also  of  ovomucoid  and  serum  globulin.  My  experimental  results 
cited  in  Chaps.  VIII  to  XI,  inclusive,  were  obtained  by  these 
methods;  they  are  obviously  applicable,  with  necessary  or  advisable 
modifications,  to  aqueous  solutions  of  the  majority  of  proteins.! 

In  the  gas-chain  determinations  two  platinized  platinum  elec- 
trodes saturated  with  hydrogen  are  used,  the  one  being  dipped 
in  the  solution  of  unknown  hydrogen-  or  hydroxyl-concentration, 
the  other  in  a  solution  of  known  hydrogen-  or  hydroxyl-concentra- 
tion, the  latter  being,  as  a  rule,  the  solution  of  acid  or  base  in  which 
the  protein  is  dissolved  and  the  former  the  solution  of  protein. 
The  electrodes  which  I  employ  are  of  a  design  due  to  my  colleague, 

*  Robertson,  T.  Brailsford,  Journ.  of  Physical  Chem.,  14  (1910),  p.  528. 
t  For  the  technique  employed  in  determining  H+  and  OH'  concentrations 
and  methods  used  in  the  computation  of  results,  consult  also 

Bovie,  W.  T.,  Journ.  Medical  Research  33  (1915),  p.  295. 

Clark,  F.  W.,  Meyers,  C.  N.,  and  Acree,  S.  F.,  Journ.  Physical  Chem.  20 

(1916),  p.  241. 

Clark,  W.  M.,  Journ.  Biol.  Chem.  23  (1915),  p.  475. 
Clark,  W.  M.,  Journ.  Biol.  Chem.  25  (1916),  p.  479. 
Clark,  W.  M.,  Journ.  Bacteriology  2  (1917),  pp.  1,  109,  191. 
Hildebrand,  J.  H.,  Journ.  Amer.  Chem.  Soc.  35  (1913),  p.  847. 
Lewis,  G.  N.,  Brighton,  T.  B.,  and  Sebastian,  R.  L.,  Journ.  Amer.  Chem.  Soc. 

39  (1917),  p.  2245. 

Michaelis,  L.,  Die  Wasserstoffionenkonzentration,  Berlin  (1914). 
Schmidt,  C.  L.  A.,  Univ.  Calif.  Publ.  Physiol.  3  (1909),  p.  101. 
Sharp,  L.  T.,  and  Hoagland,  D.  R.,  Journ.  Agric.  Research  7  (1916),  p.  123. 
Sorensen,  S.  P.  L.,  Ergeb.  d.  Physiol.  12  (1912),  p.  393. 

456 


TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS     457 

Dr.  F.  G.  Cottrell;  they  have  been  described  in  detail  by  Schmidt 
and  Finger.* 

The  hydrogen  is  generated  by  the  electrolysis  of  6  per  cent  (by 
volume)  sulphuric  acid,  in  an  apparatus  which  has  previously  been 
described  by  Schmidt  and  Finger  in  the  paper  cited  above.  To 
guard  against  the  possibility  of  any  oxygen,  ozone  or  hydrogen 
peroxide  being  carried  over  with  the  hydrogen  from  the  generator, 
the  gas  is  passed  through  a  heated  glass  tube  which  is  loosely  filled 
with  platinized  asbestos  and  which  has  wrapped  around  it  for  a 
distance  of  about  20  cm.  a  coil  of  fine  resistance- wire,  the  internal 
diameter  of  the  tube  being  about  0.5  cm.  The  hydrogen  is  com- 
pletely cooled  before  it  reaches  the  electrodes,  because,  after  leaving 
the  heater,  it  passes  through  a  narrow  glass  tube,  partly  filled  with 
glass  wool,  about  70  cm.  long,  leading  to  the  water-bath;  since  a 
considerable  portion  of  this  tube  is  within  the  incubator  which 
holds  the  water-bath,  the  temperature  of  the  hydrogen,  when  it 
reaches  the  electrodes,  may  be  considered  as  that  of  the  incubator 
itself.  The  coil  of  the  heater  attached  to  the  hydrogen-generator 
is  heated  by  a  portion  of  the  same  current  (110- volt  circuit)  which 
generates  the  hydrogen,  the  current  being  led  into  the  generator 
through  4  lamps  in  parallel,  one  of  the  lamps  being  connected  in 
series  with  the  coil.  In  order  to  maintain  the  pressure  of  hydrogen 
which  is  necessary  to  drive  it  through  the  electrodes,  the  oxygen 
which  comes  off  from  the  outer  cylinder  of  the  generator  is  carried 
off  by  a  tube  which  dips  into  a  column  of  water,  the  depth  of  the 
opening  of  the  tube  in  the  water  being  adjusted  until  the  levels  of 
the  fluid  in  the  inner  and  outer  cylinders  of  the  generator  are 
approximately  equal. 

The  complete  chain  is  arranged  as  follows:  The  syphon-tube  of 
the  "  half  -element "  containing  the  fluid  into  which  the  electrode 
dips  is  immersed  in  a  beaker  filled  with  the  same  fluid.  Thus  the 
"  half  -element "  containing  the  solution  of  unknown  hydrogen-  or 
hydroxyl-concentration  is  in  fluid  connection  with  a  beaker  which 
contains  the  same  solution  and  the  "  half-element "  containing  the 
solution  of  known  hydrogen-  or  hydroxyl-concentration  is  in  fluid 
connection  with  a  beaker  filled  with  that  solution.  The  two 
beakers  are  then  connected  by  a  U-tube  filled  with  agar  saturated 

*  C.  L.  A.  Schmidt  and  C.  P.  Finger,  Journ.  of  Physical  Chem.,  12  (1908), 
p.  406.  For  improvements  in  the  original  design,  Cf.  C.  L.  A.  Schmidt,  Univ. 
of  Calif.  Publ.  Pathol.,  2  (1916),  p.  157. 


458  APPENDIX 

with  KC1,  thus  effectively  preventing  any  mixing  of  the  two  solu- 
tions and  diminishing  any  contact-difference  of  potential  between 
them.*  The  gas  is  passed  through  the  electrode  which  dips  into 
the  solution  containing  protein  (the  solution,  that  is,  of  unknown 
hydrogen-  or  hydroxyl-concentration)  at  the  rate  of  from  one  to 
two  large  bubbles  per  second,  and  the  excess  of  gas  is  allowed  to 
pass  through  the  other  electrode.  The  whole  chain  is  immersed 
in  a  small  water-bath  which  is  placed  inside  an  incubator  main- 
tained at  a  temperature  lying  between  31  and  32  degrees  (Cf. 
below).  It  was  thought  necessary,  at  first,  not  to  permit  the 
hydrogen  to  escape  into  the  incubator,  lest  it  should  be  ignited, 
on  opening  the  door  of  the  incubator,  by  the  flame  beneath;  con- 
sequently the  electrodes  were  inserted  into  the  half-elements 
through  tightly-fitting  rubber  stoppers,  and  rubber  tubes  were 
attached  to  the  side-tubes  of  the  half-element  and  carried  outside 
the  incubator  and  the  cupboard  within  which  the  incubator  is  set 
up.  For  reasons  which  will  shortly  be  described,  however,  this 
procedure  was,  of  necessity,  abandoned  and  the  gas  was  permitted 
to  escape  into  the  incubator.  There  appears  to  be  no  danger 
involved  in  this  procedure.  The  incubator  is  of  the  usual  double- 
walled  type  employed  by  bacteriologists;  its  internal  dimensions 
are  45  cm.  wide  by  24  cm.  deep  by  48  cm.  high.  It  is  provided 
with  two  doors,  the  outer  of  the  usual  double-walled  type,  the 
inner  a  glass  door  through  which  thermometers,  etc.,  can  be  read 
without  disturbing  the  apparatus  or  causing  fluctuations  of  tem- 
perature by  currents  of  air.  The  inner  chamber  is  provided  at 
the  top  with  two  small  air-exits. 

The  potentials  between  the  electrodes  of  the  chain  are  measured 
on  a  100-cm.  potentiometer  bridge-wire  (previously  standardized).! 
For  the  detection  of  the  zero-point  on  the  bridge-wire  I  have 
employed  a  D' Arson val  galvanometer  provided  with  a  damping 
coil;  this  instrument  gave  a  decided  deflection  with  the  potential 
corresponding  to  1  mm.  displacement  on  the  bridge  in  all  of  the 
experiments  described  in  Chap.  IX.  The  constant  fall  in  potential 
from  end  to  end  of  the  potentiometer  wire  is  best  supplied  by  a 
good  storage-cell;  but  I  have  found  an  arrangement  of  four 
Gladstone-Lalande  cells  (Model  G-50),  two  in  parallel  and  two 

*  N.  Bjerrum,  Zeit.  f.  physik.  Chem.,  53  (1905),  p.  428. 
t  The  potentiometer  manufactured  by  the  Leeds  &  Northrup  Co.  is,  how- 
ever, more  convenient  to  use,  and  also  more  accurate. 


TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS     459 


in  series,  very  satisfactory.  The  potential  derived  from  these  is 
measured  against  a  standard  Weston  cell  just  before  and  just  after 
every  reading.  The  potential  provided  by  the  Gladstone-Lalande 
cells  is  quite  sufficiently  constant  during  the  progress  of  an  obser- 
vation, provided  they  are  always  short-circuited  for  15  to  20 
minutes  beforehand. 

The  electrodes  are  platinized  with  Lummer  and  Kurlbaum's 
solution.  They  are  very  carefully  washed,  both  within  and  with- 
out, between  the  observations,  first  in  a  stream  of  distilled  water 
and  then  in  the  solution  in  which  they  are  about  to  be  immersed; 
every  few  days,  if  they  are  in  constant  use,  they  should  also  be 
washed  in  chromic  and  sulphuric  acid  solution  and  then,  after 
thorough  washing  in  a  stream  of  distilled  water,  allowed  to  soak 
for  12  hours  in  distilled  water.  From  time  to  time  they  should  be 
replatinized. 

Induct  or  iiim 


Weston  Cell 


Gas  Chain 


-Hllh- 

Dry  Cells 


Short  Circuiting  Key 


,1 


Bridge  Wire 


Telephone 


Gladstone-Lalande  Cells 


The  conductivity-vessel  which  I  employ  is  of  the  Kohlrausch- 
Holborn  type,  with  a  thermometer  dipping  into  the  fluid  between 
the  electrodes.  This  is  immersed  in  the  same  water-bath  as  the 
gas-chain,  and  the  conductivities  of  the  solutions  are  measured  at 
exactly  30  degrees.  The  electrodes  are  platinized  with  Lummer 
and  Kurlbaum's  solution.  The  same  bridge-wire  is  employed  for 
determining  the  conductivities  and  for  determining  potentials.  A 
telephone  is  employed  to  detect  the  zero-point  and  the  alternating 


460  APPENDIX 

current  is  supplied  by  an  inductorium  of  the  Ostwald  type.  The 
rheostat  reads  correctly  to  within  0.01  per  cent  and  supplies  any 
desired  resistance  between  0.1  ohm  and  1000  ohms.  The  resist- 
ance in  the  rheostat  is  always  adjusted  until  the  zero-point  is 
exactly  in  the  middle  of  the  bridge;  the  resistance  in  the  rheostat 
is  then,  of  course,  exactly  equal  to  that  of  the  conductivity-vessel 
rilled  with  the  fluid  under  investigation.  The  arrangement  of  the 
wiring  is  represented  in  the  preceding  diagram. 

The  wires  of  the  conductivity  circuit  are  all  of  " bell-wire"  so  that 
their  resistance  can  be  neglected.  The  wires  of  the  potentiom- 
eter-circuit are  somewhat  thinner.  All  of  the  wires  are  insulated 
and  carefully  supported  on  glass,  and  are  never  allowed  to  touch 
the  table.  Where  it  is  necessary  to  carry  wires  through  the  table, 
for  example,  they  are  run  through  glass  tubes.  The  wire  con- 
nected with  the  slider  on  the  bridge  is  encased  in  rubber  tubing.* 

I  have  pointed  out  in  Chaps.  V  and  XII  that  it  is  difficult  to 
obtain  solutions  of  caseinates  of  much  higher  acidity  than  neutral- 
ity to  litmus  by  merely  shaking  up  casein  in  solutions  of  bases,  — 
not  because  casein  will  not  form  such  solutions,  but  because, 
although  it  dissolves  at  first  with  considerable  rapidity,  after  the 
excess  of  alkali  is  neutralized,  further  casein  dissolves  with  extreme 
slowness.  Now  the  conductivities  of  solutions  containing  proteins 
must  be  measured  as  soon  as  possible  after  the  complete  solution 
of  the  protein,  for,  otherwise,  the  hydrolysis  which  the  protein 
undergoes,  the  more  rapidly  the  more  alkaline  its  solution,  intro- 
duces a  serious  error  into  the  determination.  Hence  it  is  impera- 
tive, not  only  that  the  conductivities  of  these  solutions  should 
be  determined  as  soon  as  possible  after  complete  solution  of  the 
protein,  but,  also,  that  the  preparation  of  the  solution,  after 
the  introduction  of  the  protein,  should  consume  as  little  time  as 
possible.  Hence  all  of  the  solutions  of  casein  which  are  acid  to 
phenolphthalein  are  best  prepared  by  dissolving  the  casein  in 
excess  of  alkali  and  then  neutralizing  this  excess  with  acid. 
This  procedure,  if  gas-chain  measurements  are  being  made  at 
the  same  time,  has  the  further  advantage  that  the  absolute 
conductivities  of  the  solutions  thus  obtained  being  higher,  the 
detection  of  the  zero-point  on  the  bridge-wire  by  means  of  a  gal- 

*  More  accurate  conductance  measurements  may  be  made  by  employing 
the  method  of  E.  W.  Washburn.  Cf .  "  The  Measurement  of  Conductivity  of 
Electrolytes,"  Leeds  and  Northrup  Co.,  Philadelphia,  1916. 


TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS     461 

variometer  is  rendered  easier  and  a  less  sensitive  galvanometer 
can  be  employed  than  would  otherwise  be  required.  It  should  not 
be  forgotten,  however,  that  although  a  small  concentration  of  KC1, 
for  example,  does  not  alter  the  dissociation  of  caseinates  of  the  alka- 
lies nor  the  combining-capacity  of  casein  for  bases  (Chap.  VIII) 
yet  this  is  certainly  not  the  case  with  many  other  proteins. 

In  this  connection  it  is  to  be  carefully  noted  that  if  the  solution 
employed  to  dissolve  the  casein  be  too  alkaline,  little  or  nothing  is 
gained  by  the  rapidity  of  its  solution,  because  the  rapidity  of  its 
hydrolysis  is  also  great.  On  the  other  hand,  as  I  have  said,  if  too 
small  a  proportion  of  free  alkali  is  present  solution  is  so  slow  that 
hydrolysis  is  extensive.  Evidently  an  avoidance  of  both  of  these 
extremes  will  yield  the  most  satisfactory  results.  I  have  found 
the  proportion  10  cc.  of  N/ 10  KOH  to  1  gram  of  casein  to  be  the 
most  satisfactory  solvent  for  this  protein.  In  the  experiments 
described  in  Chaps.  IX  and  X,  save  in  the  formation  of  solutions 
containing  more  than  100  X  10~5  equivalents  of  base  per  gram  of 
casein,  therefore,  the  procedure  was  as  follows:  Part  of  the  KOH 
was  neutralized  until  the  portion  unneutralized  stood  in  this  pro- 
portion to  the  mass  of  casein  undergoing  solution,  the  casein  was 
dissolved  therein,  and  then  the  desired  final  proportion  of  KOH  to 
casein  was  attained  by  the  further  addition  of  HC1.  For  example, 
it  was  desired  to  obtain  a  solution  of  1  per  cent  casein  in  0.005 
N  KOH.  Accordingly,  to  75  cc.  of  0.1  N  KOH  were  added  50  cc. 
of  0.1  AT  HC1  and  in  this  were  dissolved  2.5  grams  of  casein;  upon 
the  attainment  of  complete  solution  and  while  stirring,  12.5  cc.  of 
0.1  N  HC1  were  added  and  the  whole  solution  was  made  up  to  250 
cc.  with  distilled  water;  another  solution  was  made  up  in  precisely 
the  same  way  but  without  the  introduction  of  the  casein;  the 
conductivities  of  both  solutions  were  than  determined  and  their 
difference  (=  X)  estimated.*  The  two  solutions  were  arranged  in 
the  gas-chain  in  the  manner  described  above  and  the  potential 
between  the  gas-electrodes  immersed  in  them  determined.  Hence, 
of  course,  the  OH'  concentration  of  the  solution  not  containing 
casein  being  known,  that  of  the  solution  containing  casein  was 

*  Since  the  concentration  of  KC1  is  the  same  in  both  solutions  it  adds  the 
same  amount  to  both  conductivities  and  this  disappears  in  their  difference; 
that  is,  presuming  that  the  caseinate  does  not  combine  with  or  decompose  the 
KC1  and  that  the  presence  of  excess  of  K+  ions  does  not  depress  the  dissociation 
of  the  potassium  caseinate;  Cf.  Chap.  VIII. 


462 


APPENDIX 


determined.  The  effect  of  the  presence  of  KC1  upon  the  dissocia- 
tion of  the  KOH  is  negligible  provided  the  KOH  and  KC1  are 
always  sufficiently  dilute  to  be  practically  completely  dissociated. 
The  extent  of  the  error  which  is  introduced  into  the  determina- 
tion of  X  by  dissolving  the  casein  in  the  first  instance  in  a  solution 
of  too  high  alkalinity  may  be  gauged  from  the  following  results : 

FINAL  SOLUTION  3  PER  CENT  CASEIN  IN  0.015  N  KOH 


Amount  of  unneutralized  KOH 
employed  to  dissolve  7.5 
grams  casein 

XX  106, 

100  cc   . 

305  0 

75  cc  .  .  .    . 

296  9 

I  have  mentioned  that  the  desired  concentration  of  the  KOH 
unneutralized  by  HC1  in  the  solutions  containing  casein  is  attained 
by  the  addition,  to  the  solutions  of  the  casein  in  excess  of  KOH,  of 
HC1  while  stirring.  This  is  a  matter  of  some  importance.  If  acid 
be  poured  into  a  solution  of  a  caseinate  which  is  imperfectly  mixed, 
the  casein  which  is  precipitated  in  the  acid  portions  of  the  fluid 
forms  bulky  coagula  and  is  only  with  difficulty  redissolved,  even 
if  the  quantity  of  alkali  still  unneutralized  by  the  acid  is  more  than 
sufficient  to  hold  in  solution  all  of  the  casein  that  may  be  present. 
Consequently,  the  solution  of  the  caseinate  must  be  rapidly  stirred 
while  the  acid  is  being  added.  The  same  procedure,  of  course, 
considerably  enhances  the  rapidity  with  which  the  casein  dissolves 
in  the  alkali  employed  for  its  solution.  I  place  the  fluid  in  which 
the  casein  is  to  be  dissolved,  together  with  the  casein,*  in  a  beaker 
of  squat  form  and  400  cc.  capacity.  The  mixture  is  then  agitated 
by  a  flattened  glass  rod  bent  at  right  angles  so  that  the  horizontal 
arm  is  about  2J  cm.  long  and  as  near  as  possible  to  the  bottom  of 
the  beaker;  this  is  rotated  at  the  rate  of  about  1600  revolutions 
per  minute  by  a  small  motor.  As  soon  as  the  casein  is  completely 
dissolved  the  acid  is  delivered  into  solution,  a  few  drops  at  a  time, 
from  a  pipette,  the  opening  of  which  is  held  at  some  depth  below  the 
surface  of  the  liquid.  In  some  of  the  earlier  experiments  the  acid 
was  poured  upon  the  surface  of  the  solution,  but  all  of  these  solu- 
tions foam  to  a  certain  extent  and  the  foam  is  not  agitated  by  the 

*  Regarding  the  procedure  to  be  followed  in  adding  the  casein  to  the  solvent 
Cf.  Chap.  XII,  1. 


TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS     463 

stirrer  with  the  same  rapidity  as  the  fluid  which  lies  below  it, 
consequently,  if  the  acid  is  poured  upon  the  surface,  casein  is 
precipitated  within  the  foam  and  is  only  with  great  difficulty 
redissolved,  hence  solutions  prepared  in  this  manner  yielded  very 
irregular  results  both  in  the  gas-chain  and  in  the  conductivity  de- 
terminations. In  delivering  the  acid  into  the  solution  it  is  very 
necessary  to  avoid  holding  the  opening  of  the  pipette  too  close  to 
the  side  of  the  beaker,  as  in  that  case  a  film  of  casein  is  precipitated 
on  the  glass  and  this  film  redissolves  with  great  difficulty. 

Since  the  conductivities  of  solutions  of  the  caseinates  must  be 
determined  as  soon  as  possible  after  the  introduction  of  casein 
into  the  solution  employed  to  dissolve  it,  for  otherwise  hydrolysis 
introduces  a  considerable  error,  it  is  evident  that  as  little  time  as 
possible  must  be  consumed  in  bringing  the  temperature  of  the 
solution  to  that  at  which  its  conductivity  is  to  be  determined. 
This  could  be  achieved  in  either  of  two  ways:  either  a  small 
volume  of  fluid  could  be  employed,  so  disposed  as  to  take  up  the 
temperature  of  the  water-bath  very  quickly;  or  the  water-bath 
may  be  maintained  at  a  somewhat  higher  temperature  than  that 
actually  desired,  and  the  conductivity  of  the  fluid  can  be  measured 
at  the  moment  when  it  reaches  the  desired  temperature.  For 
reasons  which  will  be  sufficiently  obvious  the  latter  procedure  is 
found  to  be  more  convenient.  The  water-bath  is  kept  at  a  tem- 
perature lying  between  31.5  and  32.5  degrees,  a  preliminary 
measurement  of  the  conductivity  of  the  solution  is  made  at  a 
temperature  between  29  and  29.5  degrees  and  this  preliminary 
determination  is  corrected  at  precisely  30  degrees.* 

Solution  of  1  per  cent  casein  in  0.03  N  KOH : 

X  X  105  determined  immediately 348. 8 

X  X  105  determined  after  allowing  solution  to  stand  at  about 

30  degrees  for  20  minutes 356. 3 

It  is  obvious,  however,  that  in  this  procedure  the  gas-chain 
measurements  are  made  at  a  temperature  some  2  degrees  higher 
than  the  conductivity  measurements,  and  it  may  be  inquired  to 

*  For  maintaining  constant  temperatures,  W.  M.  Clark  employs  an  air- 
bath;  Cf.  Journ.  Amer.  Chem.  Soc.,  35  (1913),  p.  1889;  Journ.  Biol.  Chem., 
23  (1915),  p.  475.  C.  L.  A.  Schmidt,  Univ.  of  Calif.  Publ.  Pathol.,  2  (1916), 
p.  157,  employs  an  oil-bath  similar  in  construction  to  that  described  by 
G.  A.  Hulett,  Physical  Review,  32  (1911),  p.  257. 


464  APPENDIX 

what  extent  this  invalidates  the  comparison  of  the  two  sets  of 
determinations.  The  error  which  is  thus  introduced,  for  the  small 
difference  of  temperature  concerned  is,  however,  negligible,  since 
it  was  found  by  actual  trial  that  the  difference  between  the  poten- 
tials measured  at  30  degrees  and  those  measured  at  34  degrees 
could  barely,  with  certainty,  be  detected  upon  my  potentiometer- 
bridge.  Nor  is  this  fact  surprising  for,  exclusive  of  any  possible 
change  in  the  equilibrium  between  protein  and  alkali,  the  potential, 
according  to  the  Nernst  formula,  varies  directly  as  the  absolute 
temperature,  and,  therefore,  only  increases  by  1 /300th  per  degree 
centigrade  at  30  degrees.  It  has  been  observed  by  W.  A.  Osborne* 
and  myself  f  that  there  is  no  evidence  of  an  appreciable  shift  in  the 
equilibrium  between  casein  and  alkali,  as  the  temperature  rises, 
until  the  temperature  of  36  degrees  is  reached. 

I  have  mentioned  in  describing  the  apparatus  employed,  that 
difficulty  was  encountered  in  leading  off  the  hydrogen,  after  it  had 
bubbled  through  the  fluids,  through  the  exit-tubes  of  the  half- 
element  to  the  outside  of  the  incubator.  The  difficulty  was  this, 
—  all  of  the  solutions  containing  casein  foam  upon  passing  the 
hydrogen  through  them,  and  to  a  greater  extent  the  less  the  excess 
of  alkali  in  the  solutions.  This  foam,  collecting  in  the  exit  and 
connected  rubber  tubes,  gave  rise  to  a  pressure  which  drove  the 
fluid  out  of  the  half-element  and  thus  interrupted  the  continuity 
of  the  chain.  Accordingly,  it  was  found  necessary,  not  only  to 
abandon  the  idea  of  conveying  the  waste  hydrogen  out  of  the 
incubator,  but  also  to  cut  off  short  the  exit-tube  of  the  half-element 
and  permit  the  foam  to  escape  freely  into  the  water-bath  (the 
lower  end  of  the  exit-tube  was,  of  course,  well  above  the  surface  of 
the  water  in  the  bath).  After  this  plan  had  been  adopted  no 
further  trouble  was  encountered  from  this  source.  Foaming,  may, 
however,  be  prevented  or  greatly  reduced  by  the  addition  of  a  few 
drops  of  octyl  or  amyl  alcohol. 

All  observers  who  have  endeavored  to  measure  the  conductivi- 
ties of  solutions  containing  proteins  have  encountered  the  difficulty 
involved  in  the  precipitation  which  occurs  at  the  electrodes, 
particularly  in  the  case  of  casein,  in  neutral  or  very  faintly  acid 
solutions.  Whetham  and -Hardy  {  in  order  to  minimize  the  error 

*  W.  A.  Osborne,  Journ.  Physiol.,  27  (1901),  p.  398. 

t  T.  Brailsford  Robertson,  Journ.  Biol.  Chem.,  5  (1908),  p.  147. 

t  W.  B.  Hardy,  Journ.  of  Physiol.,  33  (1905),  p.  251. 


TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS     465 

arising  from  this  source,  adopted  the  plan  of  heating  the  electrode, 
after  platinization,  to  a  dull  red,  thus  clumping  the  platinum  black 
and  reducing  the  total  surface  of  the  electrode. 

I  formerly  tacitly  assumed  this  phenomenon  to  be  connected  in 
some  way  with  the  passage  of  the  alternating  current  through  the 
solution.  In  my  own  experiments,  however,  I  speedily  found  that 
in  neutral  or  faintly  acid  solutions  of  potassium  caseinate  marked 
precipitation  of  casein  occurs  at  the  gas  electrode,  without  the 
passage  of  any  current,  usually  after  but  sometimes  even  before 
the  passage  of  the  hydrogen.  At  first  I  was  inclined  to  attribute 
this  to  gases  collected,  in  the  interval  between  experiments,  in  the 
tubes  conveying  the  hydrogen,  to  impurities  in  the  hydrogen,  etc., 
—  but  after  very  careful  exclusion  of  all  of  these  possibilities  the 
precipitation  still  took  place,  and  it  occurred  to  me  that  it  might 
be  due  to  the  hydrogen  ions  dissolved  in  the  platinum  itself. 
When  hydrogen  is  passed  through  a  platinized  platinum  electrode, 
the  potential  measured  across  the  chain  represents,  at  first,  a 
higher  acidity  of  this  electrode  than  it  does  later  on,  when  the 
electrode  has  come  into  equilibrium  with  the  solution;  in  other 
words,  the  hydrogen  ions  dissolved  in  the  platinum  have  not  yet 
come  into  equilibrium  with  those  in  the  solution;  an  excess  of 
hydrogen  ions  is  still  present  in  the  platinum.  It  therefore 
appeared  possible  that  this  initial  acidity  of  the  platinum  itself 
might  be  responsible  for  the  precipitation  of  casein  at  its  surface, 
and  this  idea  found  confirmation  in  the  fact  that  on  prolonged 
passage  of  hydrogen  the  film  of  protein  deposited  on  the  electrode 
slowly  redissolved.  It  occurred  to  me  that  it  might  be  possible  to 
avoid  this  precipitation  altogether  by  bringing  the  electrode  nearly 
to  equilibrium  with  a  low  concentration  of  hydrogen  ions  before 
introducing  it  into  the  solution  at  all;  accordingly,  before  making 
a  gas-chain  determination  with  a  caseinate  solution  of  very  low 
hydroxyl  concentration  (neutral  or  acid  to  litmus)  the  electrode 
which  was  to  be  dipped  into  the  protein  solution  was  immersed  in 
distilled  water  and  gas  was  passed  through  it  for  an  hour  or  more  — 
it  was  then  immediately  washed  in  the  protein  solution  and  used. 
The  device  was  found  to  work  excellently,  and  in  all  of  the  experi- 
ments described  herein  precipitation  at  the  electrodes  was  avoided 
entirely,  at  least  so  far  as  the  eye  could  perceive.  A  similar  device 
was  found  effective  in  avoiding  precipitation  at  the  electrodes  of 
the  conductivity-vessel.  The  vessel  was  simply  filled  with  dis- 


466  APPENDIX 

tilled  water  and  allowed  to  stand  in  the  water-bath  for  some  hours 
before  making  a  determination. 

In  order  to  ensure  a  correct  determination  of  the  potential  of  the 
chain  in  these  experiments  it  was  always  found  necessary  to  pass 
the  gas  for  three  hours  and,  in  the  neutral  or  faintly  acid  solutions, 
for  as  much  as  six  hours.  If  two  successive  readings  on  the  bridge, 
taken  an  hour  apart,  did  not  differ  by  more  than  1  mm.  the  result 
was  considered  correct.  Difficulty  in  obtaining  constant  readings 
is,  of  course,  only  encountered  in  the  chains  yielding  the  highest 
potentials,  in  which,  as  the  apparatus  is  arranged,  1  mm.  on  the 
bridge-wire  makes  a  difference  of  only  about  one-half  per  cent  to 
the  calculated  value  of  the  potential.  In  the  chains  of  low  poten- 
tial greater  accuracy  is  desired,  but  no  difficulty  is  encountered  in 
attaining  it,  since,  in  these,  successive  readings  are  nearly  always 
identical,  or  if  they  are  not,  the  difference  can  invariably  be  traced 
to  some  obvious  source  of  error  which  may  be  eliminated  in  a 
repetition  of  the  experiment. 

Since,  however,  prolonged  exposure  of  the  protein  solution  in 
the  gas-chain  to  the  temperature  of  the  water-bath  is  essential,  the 
question  arises  whether  the  accuracy  of  the  determinations  is  not 
invalidated  by  hydrolysis  of  the  casein.  The  answer  to  this  ques- 
tion is  in  the  negative,  the  change  in  the  hydrogen-  or  hydroxyl- 
concentration  of  a  protein  solution,  due  to  hydrolysis,  is  negligible 
in  comparison  with  the  change  in  its  conductivity.  Moreover,  it 
has  been  shown*  that  the  displacement  of  the  neutral  point  on  the 
potentiometer-wire,  due  to  hydrolysis  of  the  casein,  in  an  alkaline 
solution  of  a  caseinate  is  opposed  in  sense  to  the  displacement  due 
to  the  coming  to  equilibrium  of  the  electrode  with  the  solution  in 
which  it  is  dipped.  Were  appreciable  change  in  the  hydroxyl 
concentration  of  alkaline  solutions  of  casein  due  to  hydrolysis 
occurring,  therefore,  the  position  of  the  neutral  point  on  the  bridge 
should  indicate  at  first  a  diminishing  and  later  increasing  potential. 
I  have  discovered  no  trace  of  this  save  in  one  or  two  of  the  most 
alkaline  solutions  which  I  have  employed  (1  per  cent  casein  in 
0.03  NKOH;  3  per  cent  casein  in  0.05  N  KOH),  in  these  the 
displacement  due  to  hydrolysis  was  never  more  than  1  mm.  and 
the  minimum  value  of  the  potential  was  taken  as  the  true  one.  It 
is,  however,  to  be  recollected  that  the  hydrolysis  was  probably 

*  T.  Brailsford  Robertson  and  C.  L.  A.  Schmidt,  Journ.  Biol.  Chem.,  5 
(1908),  p.  31. 


TECHNIQUE  OF  ELECTROCHEMICAL  MEASUREMENTS     467 

most  rapid  in  the  period  of  time  preceding  the  attainment  of  this 
minimum,  so  that  even  the  minimum  potential  may  be  consider- 
ably in  error,  especially  as  in  the  chains  containing  these  solutions 
the  potential  was  low  and  1  mm.  displacement  on  the  bridge 
introduced  a  considerable  error  into  the  determination  of  the 
potential.  This  error  is  diminished  in  its  percentage  magnitude 
in  the  calculation  therefrom  of  m,  the  amount  of  alkali  neutralized 
by  the  protein,  but,  nevertheless,  the  determination  of  m  in  the 
solutions  mentioned  is  not  to  be  considered  trustworthy.  In  less 
alkaline  solutions  no  effect  upon  the  hydroxyl-concentration  of 
the  caseinate  solutions,  due  to  hydrolysis,  could  be  discovered; 
had  such  an  effect  been  present  to  any  appreciable  extent  it  would, 
of  course,  have  been  detected  in  these  solutions  much  more  readily 
than  in  those  in  which  such  an  effect  was  detected,  since  the  poten- 
tials of  the  chains  containing  these  solutions  were  higher,  so  that 
a  smaller  change  in  hydroxyl-concentration  of  the  solution  would 
have  produced  a  greater  (absolute)  displacement  of  the  neutral 
point  upon  the  bridge-wire. 

In  concluding  these  remarks,  it  may  be  stated  that  unless  all  of 
the  precautions  which  have  been  described  are  observed  with  the 
utmost  fidelity  the  results  of  experiments  such  as  these,  and  con- 
ducted in  this  manner,  will  be  found  to  be  wholly  irregular,  and, 
save  in  a  qualitative  sense,  untrustworthy. 


INDEX  OF  AUTHORS 


The  numbers  in  heavy  type  refer  to  the  bibliographies. 


ABDERHALDEN,  E.,  31,  32,  161,  387, 
388,  15,  16,  154,  343,  371,  372,  373, 
374,  375,  376,  377,  378,  379,  381, 
382,  384,  385. 

ABEGG,  R.,  217,  195. 

ACHALME,  P.,  453,  449. 

ACRES,  S.  F.,  420,  395,  456. 

ALEXANDER,  A.  C.,  367,  357,  358. 

ALEXANDROV,  N.,  353,  331. 

ALLEMANN,  O.,  105,  161,  91,  148. 

ALSBERG,  C.  L.,  350,  339. 

ALTMANN,  R.,  161,  143. 

AMMANN,  L.,  357. 

ANDERSON,  H.  C.,  420,  398. 

ANDERSON,  J.  A.,  135,  126. 

ANNENKOV,  A.,  33,  6. 

ANSIAUX,  G.,  317,  308. 

ARMSTRONG,  E.  F.,  421,  453,  393,  425. 

ARNY,  H.  V.,  65,  105,  56,  97. 

ARRHENIUS,  S.,  161,  271,  350,  420, 
156,  189,  266,  306,  320,  341,  342, 
402,  403,  404,  419,  420. 

BABO,  P.,  134,  126. 

BANG,  I.,  420,  418. 

BARCROFT,  J.,  161,  350,  160,  161,  337. 

BARTH,  L.,  31,  4. 

BASSET,  H.,  134,  127. 

BAYLISS,  W.  M.,  83,  350,  388,  420, 

453,  80,  340,  369,  402,  403,  435,  447, 

449,  450. 

BEARN,  A.  R.,  453,  449. 
BEATTY,  W.  A.,  32,  16. 
BECHAMP,  A.,  105,  90. 
BECHHOLD,  H.,  161,  350,  143,  322. 
BEERS,  W.  H.,  84,  82. 
BELL,  J.  M.,  317,  285. 
BENEDICENTI,  A.,  161,  350,  142,  349. 
BERCZELLER,  L.,  161,  316,  350,  148, 

310,  345. 

BERG,  W.  N.,  420,  410,  411. 
BERGELL,  P.,  387,  388,  15,  371,  374. 
BERNARD,  C.,  134,  108. 


BERNINZONE,  420,  453,  393,  425. 
BERTHELLOT,  M.  P.  E.,  350,  330. 
BERTHOLD,  G.,  350,  346. 
BERZELIUS,  J.,  420,  391. 
BEUTNER,  R.,  194,  316,  193,  299. 
BIDDLE,  H.  C.,  423,  454,  393,  435,  437. 
BIEN,  Z.,  218,  210. 

BlERNACKE,  E.,  83,  80. 

BIGLAND,  D.,  352,  337. 

BILLITZER,  J.,  31,  134,  26,  108,  118. 

BILTZ,  W.,  134,  119. 

BlRCHARD,  F.  J.,  33,    19. 

BJERRUM,  N.,  217,  197,  458. 

BLACK,  O.  F.,  218,  214. 

BLASEL,  L.,  31,  54,  194,  217,  23,  51, 

168,  204. 

BLISH,  M.  J.,  32,  17. 
BLUM,  F.,  161,  156,  157,  158. 
BODENSTEIN,  M.,  420,  453,  393,  425. 
BOEHM,  R.,  423,  415. 
BOGDANDY,  S.,  420,  405. 
BOGUSKY,  J.  G.,  316,  277. 
BOHR,  C.,  162,  159,  160.       . 
BONAMARTINI,  G.,  134,  162,  133,  140. 
BORISSOV,  P.,  420,  403. 
BORKEL,  C.,  367,  356,  358. 
BOSWORTH,  A.  W.,  54,  66,  105,  106, 

251,  420,  424,  37,  42,  56,  89,  90,  91, 

92,  93,  98,  101,  235,  249,  418,  419. 
BOTTAZZI,  F.,  317,  350,  351,  299,  320, 

322,  345. 

BOTTGER,  W.,  83,  78. 
BOVIE,  W.  T.,  317,  310,  456. 
BOWERS,  W.  G.,  83,  78. 
BRADLEY,  H.  C.,  317,  420,  453,  316, 

392,  393,  425,  427,  452,  453. 
BRAEUNNING,  H.,  420,  415. 
BRAHM,  C.,  387,  385. 
BREDIG,  G.,  31,  83, 134, 194,  250,  388, 

26,  67,  108,  118,  184,  233,  235,  369. 
BRIDGMAN,  P.  W.,  317,  310. 
BRIGGS,  R.  S.,  65,  65. 
BRINK,  F.  N.,  351,  329. 


469 


470 


INDEX  OF  AUTHORS 


BRITO,  P.  S.,  194,  177,  190. 
BBOECK,  C.  T.,  31,  30. 
BROOK,  F.  W.,  162,  157,  158. 
BROWN,  A.  P.,  55,  318,  319,  44,  311, 

313,  314. 

BROWNING,  C.  H.,  162,  155. 
BRUNER,  L.,  317,  276. 
BRUNNER,  E.,  317,  277. 
BRUYN,  LOBRY  DE,   M.  C.  A.,  351, 

342. 

BUBNOW,  N.  A.,  420,  415. 
BUCHNER,  E.,  375. 
BUCK,  L.  W.,  65,  65. 
BUGARSZKY,  S.,  32,  83,  134,  194,  217, 

351,  26,  75,  76,  77,  78, 134, 167, 170, 

195,  332. 

BUGLIA,  G.,  317,  351,  304,  345. 
BULOW,  K,  367,  358. 
BUNGE,  G.  VON,  217,  213. 
BURIAN,  R.,  105,  86. 
BURNETT,  T.  C.,  33,  251,  271,  353, 

28,  237,  238,  253,  333,  339. 
BURTON,  D.,  319,  293,  298. 
BUTTERFIELD,  E.  E.,  162,  317,  367, 

161,  313,  358. 
BUTTNER,  C.,  422,  389. 

CAEMMERER,  G.,  387,  385. 
CAMERON,  A.  T.,  32,  163,  22,  158. 
CAMERON,  K.  F.,  317,  285. 
CAMIS,  M.,  161,  160. 
CAMPBELL,  G.  F.,  54,  163,  368,  44, 

148,  355,  356. 

CATHCART,  E.  P.,  453,  449. 
CESANA,  G.,  420,  420. 
CHAIN,  A.,  454,  438. 
CHIARI,  R.,  54,  317,  51,  293,  298. 
CHICK,  H.,  83,  134,  317,  351,  70,  108, 

123,  129,  305,  307,  308,  309,  328, 

349. 
CHITTENDEN,  R.  H.,  83, 135,  368,  420, 

454,  80,  109,  355,  357,  415,  438.  / 
CHRISTIANSEN,  J.,  351,  328.  / 
CLAPP,  S.  H.,  33,  16. 
CLARK,  F.  W.,  456. 
CLARK,  W.  M.,  84,  81,  456,^63. 
CLEMENT,  M.,  420,  392. 
COGGI,  454,  449. 
COHEN,  E.,  271,  262. 
COHNHEIM,  O.,  83,  105,  162,  71,  79, 

80,  88,  138. 

CONROY,  J.  T.,  420,  395. 
COOKE,  E.,  317,  299. 


COPPADORE,  A.,  420,  397. 
CORIN,  J.,  317,  308. 
CORRAL,  J.  M.  DE,  217,  211. 
COTTRELL,  F.  G.,  294,  457. 
COURANT,  G.,  105,  89,  90. 
CRAMER,  M.,  420,  453,  393,  449. 
CUMMINGS,  A.  C.,  217,  197. 
CUMMINS,  A.  W.,  420,  415. 
CURTIUS,  T.,  32,  9. 

DABROVSKY,  S.,  351,  330,  331,  338. 
D'AGOSTINO,  E.,  83,  217,  351,  78,  204, 

322 

DAKIN,  H.  D.,  32,  194,  30,  31,  190. 
DALE,  T.  P.,  367,  362. 
DAM,  W.  VAN,  194,  424,  169,  418. 
DANEEL,  H.,  351,  326. 
D'ARSONVAL,  A.,  367,  358. 
DASTRE,  A.,  453,  449. 
DAUWE,  F.,  420,  399. 
DEMPWOLFF,  C.,  351,  326. 
DERHAM,  H.  G.,  134,  127. 
DBS  BANCELS,  L.,  421,  397,  402,  403. 
DESORMES,  M.,  420,  392. 
DIETZ,  W.,  420,  453,  393,  425,  451. 
DONATH,  H.,  453,  449. 
DONNAN,  F.  G.,  134,  127,  128. 
DRECHSEL,  E.,  32,  162,  5,  158. 
DUCLAUX,  J.,  351,  340. 
DUDLEY,  H.  W.,  32,  30. 
DUHEM,  P.,  351,  341. 
DUMANSKI,  A.,  351,  323,  325. 

EDKINS,  J.  S.,  83,  80. 

EHRENBERG,  R.,  317,  293,  297. 

EHRLICH,  P.,  449. 

EINSTEIN,  A.,  331. 

ELY,  J.  S.,  83,  80. 

EMMERLING,  O.,  421,  453,  393,  425. 

ERB,  W.,  32,  83,  217,  21,  72,  78,  206. 

ERNST,  C.,  421,  392. 

EULER,  H.,  388,  421,  453,  369,  383, 

384,  392,  393,  396,  444,  445,  449. 
EVES,  F.,  83,  80. 
) 

FALEK,  O.,  163,  148. 
FALK,  F.,  83,  80. 
FARADAY,  M.,  351,  349. 
FARKAS,  G.,  217,  218,  211. 
FARMER,  J.  B.,  317,  307. 
FEINSCHMIDT,  J.,  162,  148. 
FELS,  B.,  83,  81,  212. 
FERNAN,  A.,  317,  310. 


INDEX  OF  AUTHORS 


471 


FERNET,  E.,  218,  213. 

FICK,  A.,  361,  330. 

FIELD,  C.  W.,  66,  58,  60. 

FINDLAY,  A.,  134,  123. 

FINGER,  C.  P.,  78,  457. 

FISCHER,  E.,  32,  388,  421,  5,  10,  11, 

12,  13,  15,  18,  23,  371,  372,  373, 

374,  393. 

FISCHER,  M.  H.,  317,  299. 
FISCHER,  W.,  453,  444. 
FOA,  C.,  218,  212. 
FOURNEAU,  E.,  32,  10,  11. 
FRAENKEL,  P.,  218,  211. 
FRAMM,  F.,  367,  358. 
FRANKEL,  E.  M.,  421,  398. 
FRASER,  J.  W.,  421,  415. 
FREDERICQUE,  L.,  317,  367,  305,  355, 

356. 

FREI,  W.,  361,  345. 
FREUND,  E.,  54,  40. 
FREUNDLICH,  H.,  134,  108,  117,  118, 

291. 
FRIEDENTHAL,  H.,  83,  218,  81,  212, 

214. 
FULD,  E.,  162,  421,  143,  416,  417,  419, 

420. 

FUNK,  C.,  421,  418. 
FURTH,  O.  VON,  162,  158. 

GALEOTTI,  G.,  134, 162,  108,  121,  124, 

125,  138,  139,  275. 
GAMGEE,  A.,  351,  367,  349,  356,  358. 
GANSSER,  E.,  162,  160. 
GARRETT,  H.,  351,  327,  328. 
GAVRILOV,  N.,  32,  19. 
GAY,  F.  P.,  162,  421,  453,  149,  154, 

155,  393,  436,  437. 
GEAKE,  A.,  421,  418. 
GERBER,  C.,  421,  417. 
GEVIN,  J.  W.  A.,.  421,  418. 
GIBBS,  J.  W.,  134,  123,  345. 
GIBSON,  R.  B.,  317,  310. 
GIES,  W.  J.,  164,  420,  146,  410,  411. 
GIGON,  A.,  387,  376,  384,  385. 
GILLESPIE,  A.  L.,  421,  411. 
GJALDBAEK,  J.  K.,  421,  463,  398,  437. 
GLADSTONE,  J.  H.,  367,  362,  363,  366, 

367. 

GOKUM,  F.,  351,  327. 
GOMBERG,  M.,  194,  191. 
GORTNER,  R.  A.,  32,  17. 
GOTO,  M.,  106,  86,  87. 
GRAHAM-STEFAN,  330. 


GRAHAM,  T.,  134,  162,  351,  112,  142, 

143,  322,  330,  337,  342. 
GRAMENISKY,  M.  Y.,  463,  450. 
GRAVES,  S.  S.,  66,  58. 
GREAVES,  J.  E.,  368,  48,  359,  361, 

365,  366. 

GREEN,  W.  H.,  351,  323. 
GREENWOOD,  M.,  421,  411. 
GRIMEAUX,  E.,  32,  9. 
GRINEFF,  W.,  218,  210. 
GROSS,  O.,  421,  403,  405. 
GRUEBLER,  G.,  54,  48. 
GUTHE,  K.  E.,  194,  178. 

HAEBLER,  M.,  361,  348. 

HAHN,  M.,  453,  454,  432,  449. 

HALLIBURTON,  W.  D.,  317,  305,  314, 
315. 

HAMBURGER,  H.  J.,  83,  218,  76,  195. 

HAMMARSTEN,  O.,  54,  105,  361,  421, 
37,  40,  41,  93,  343,  400,  418. 

HANDOVSKY,  H.,  135,  317,  122,  131, 
305. 

HANRIOT,  M.,  421,  453,  393,  425. 

HARDNACK,  E.,  162,  158. 

HARDY,  W.  B.,  64,  83,  105,  134,  162, 
194,  218,  250,  317,  351,  364,  40,  74, 
75,  76,  80,  98,  99,  100,  107,  108,  113, 
114,  117,  124,  131,  134,  150,  152, 
153,  171,  176,  187,  188,  189,  190, 
205,  226,  227,  230,  250,  275,  300, 
301,  302,  303,  304,  320,  322,  323, 
328,  329,  333,  464. 

HARI,  P.,  317,  453,  308,  451. 

HARRIS,  F.  I.,  367,  359. 

HARRIS,  I.  F.,  55,  48,  357. 

HARRIS,  V.  D.,  421,  415. 

HART,  E.  B.,  65,  84,  106,  219,  424,  40, 
69,  89,  91,  93,  95,  97,  204,  207,  418. 

HARTLEY,  W.  N.,  134,  135,  126,  127. 

HASSELBALCH,  K.  A.,  218,  211. 

HAYCRAFT,  J.  B.,  351,  329. 

HAYWOOD,  J.  K.,  105,  95. 

HEDBLOM,  C.  A.,  360,  339. 

HEDIN,  S.  G.,  32,  162,  421,  453,  5, 
156,  399,  404,  418,  449. 

HEIDENHAIN,  M.,  83,  162,  82,  146. 

HEIDENHAIN,  R.,  218,  421,  213,  415. 

HEKMA,  E.,  105,  101. 

HEMMETER,  J.  C.,  421,  418. 

HENDERSON,  L.  J.,  218,  317,  361, 
453,  214,  215,  216,  308,  329,  451. 

HENDRIX,  B.  M.,  33,  31. 


472 


INDEX  OF  AUTHORS 


HENRI,  V.,  388,  421,  380,  382,  384, 

397,  402,  403,  406,  407,  408. 
HENEIQUES,  V.,  421,  453,  398,  437. 
HENSEL,  454,  449. 
HENZE,  M.,  162,  142,  157,  158. 
HEBLITZKA,  A.,  367,  359,  366,  367. 
HERMANN,  L.,  351,  346. 
HERRMANN,  J.,  454,  438. 
HERTZ,  A.  F.,  352,  347. 
HERZOG,  R.  O.,  351, 352, 421, 330, 418. 
HEUBNER,  W.,  317,  367,  313,  358. 
HEYDWEILLER,  A.,  218,  204,  215. 
HILDEBRAND,  J.  H.,  83,  78,  456. 
HILL,  A.  V.,  161,  350,  160,  337. 
HILL,  CROFT,  421,  454,  356,  393,  425, 

427. 

HIRSCHFELD,  M.,  33,  219,  23,  203. 
HIRSCHSTEIN,  L.,  164,  194,  140,  170. 
HOAGLAND,  D.  R.,  456. 
HOBER,  R.,  135,  218,  120,  211. 
HOFF,  VAN'T,  123, 160,  306,  308,  420. 
HOFMEISTER,  F.,  32, 135, 162,  317, 18, 

21,  22,  23,  108,  109,  110,  111,  125, 

157,  292,  299,  309,  311,  339. 
HOLBORN,  L.,  194,  174. 
HOLZBERG,  H.  L.,  162,  146. 
HOPKINS,  F.  G.,  54, 162,  318,  367,  44, 

157,  158,  311,  355. 
HOPPE-SEYLER,  F.,  54,  162,  218,  367, 

45,  148,  214,  355,  356,  358. 
HOWELL,  W.  H.,  194,  318,  421,  422, 

454,  177,  190,  316,  416,  419,  450. 
HOYT,  H.  S.,  367,  359. 
HUFNER,  G.,  32,  162,  318,  352,  367, 

4,  159,  160,  313,  322. 
HULETT,  G.  A.,  352,  347,  463. 

HUNDESCHAGEN,  F.,  162,   158. 

HUNTER,  A.,  162,  387,  148,  149. 
HUPPERT,  H.,  423,  403,  404. 
HURWITZ,  S.  H.,  65,  65. 

ISCOVESCO,  H.,  352,  345. 
IZAR,  G.,  162,  148. 

JACOBY,  M.,  422,  418. 
JAGER,  L.  DE,  105,  89. 
JAMISON,  R.,  352,  347. 
JAQUET,  A.,  162,  160. 
JESSEN-HANSEN,  H.,  33,  19,  22. 
JEWETT,  R.  M.,  65,  65. 
JOACHIM,  J.,  54,  40. 
JONES,  H.  C.,  135,  271,  352,  126,  127, 
130,  266,  323. 


JONES,  W.,  367,  356. 
JURGENSEN,  E.,  319,  309. 

KAJANDER,  N.,  317,  277. 
KANITZ,  A.,  422,  411. 
KASERNOVSKY,  H.,  351,  352,  330. 
KASTLE,  J.  H.,  422,  454,  393,  425. 
KATZ,   J.   R.,    318,    292,    311,    315, 

316. 

KAUDER,  G.,  135,  109. 
KAUFFMANN,  R.,  422,  415. 
KELLERSBERGER,  E.,  453,  452. 
KENDALL,  E.  C.,  163,  158. 
KENNAWAY,  E.  L.,  163,  158. 
KING,  W.  O.  R.,  161,  160. 
KIRSCHOFF,  J.,  422,  392. 
KJELDAHL,  J.,  357. 
KLUG,  F.,  422,  410. 
KOBER,  P.  A.,  32,  65,  163,  352,  367, 

22,  30,   31,  58,  59,  60,   142,   345, 

359. 
KOELKER,  A.  H.,  31,  387,  388, 15,  376, 

377,  382,  385. 
KOENIGS,  E.,  32,  23. 
KOHLRAUSCH,  F.,  194,  218,  174,  204, 

215. 

KONOVALOV,  D.,  352,  342. 
KOPP,  H.,  352,  343. 
KOROSY,  K.,  318,  299. 
KOSSEL,  A.,  32,  54,   105,  163,  250, 

368,  5,  8,  19,  21,  22,  25,  30,  53,  54, 

86,  87,  143,  148,  158,  235,  241,  242, 

363. 

KOSUTANY,  T.,  54,  49. 
KOUKOL-YASNOPOLSKY,  W.,  422,  389. 
KRAFFT,  F.,  352,  332. 
KRAUSE,  E.,  164,  156. 
KRIEGER,  H.  T.,  83,  318,  71,  311. 
KRONIG,  B.,  83,  82. 
KRUGER,  T.  H.,  368,  356. 
KRUKENBERG,  422,  389. 
KUHNE,  W.,  54, 135,  368,  454,  41, 109, 

355,  374,  438. 
KULIKOV,  J.,  33,  6. 
KURAJEV,  D.,  105,  454,  87,  438. 
KUTSCHER,  F.,  32,  163,  5,  148. 

LACQUBUR,  E.,  54,  105,  218,  422,  40, 

88,  89,  204,  207,  418. 
LA  FRANCA,  S.,  83,  82. 
LANDOLT,  H.,  368,  362. 
LANDSTEINER,  K.,  454,  449. 
LANGLEY,  J.  N.,  83,  80. 


INDEX  OF  AUTHORS 


473 


LANGSTEIN,  L.,  161,  154. 
LARMOR,  J.,  362,  341. 
LAVROV,  D.,  454,  438. 
LEAVBNWORTH,  C.  S.,  33, 83, 105, 163, 

19,  24,  71,  103,  142. 
LE  CHATELIER,  H.  L.,  135,  126. 
LEHMANN,  J.,  352,  346. 
LEICK,  A.,  352,  329. 
LENGYEL,  R.  VON,  318,  454,  308,  451. 
LENK,  E.,  318,  293. 
LESCOEUR,  H.,  105,  95. 
LEVENE,  P.  A.,  32,  16. 
LEVITES,   S.,  32,   352,    19,   20,  320, 

327. 

LEWIN,  M.,  135,  126. 
LEWIS,  G.  N.,  135,  352,  126,  127,  326, 

456. 

LEWITH,  I.,  135,  109. 
LEY,  H.,  218,  206. 
LICHTWITZ,  L.,  352,  328. 
LIEBERMANN,  L.,  32,  83, 134, 163, 194, 

217,  351,  26,  75,  76,  77,  78,  134, 

143,  167,  170,  195,  332. 
LIEBIG,  J.,  32,  4,  391. 
LIEBRECHT,  A.,  163,  157. 
LIEBREICH,  O.,  352,  347. 
LIESEGANG,  R.  E.,  318,  304. 
LILLIE,  R.  S.,  352,  337,  338,  339. 

LlLIENFELD,  L.,  163,  146. 
LlNDBERGER,  V.,  422,  415. 

LINDER,  S.  E.,  135,  112, 113, 114,  115, 

116,  117. 
LINDET,  L.,  357. 

LlNEBARGER,  C.  S.,  352,  342. 

LIPPICH,  F.,  163,  141. 

LODGE,  O.,  352,  323. 

LOEB,  J.,  83,  135,  194,  218,  318,  422, 

82,  118,  193,  212,  214,  299,  313, 

410,  411. 

LOEB,  L.,  422,  416. 
LOEVENHART,  A.  S.,  105,  194,  422, 

454,  91,  97,  169,  393,  425. 
LOEW,  O.,  163,  194,  158,  193. 
LOHNSTEIN,  T.,  352,  348. 
LOMBARDI,  M.,  134,  162. 
LORENTZ,  H.  A.,  368,  367. 
LORENTZ,  L.,  368,  367. 
LUBAVIN,  N.,  422,  464,  389,  435. 
LUBRZYNSKI,  E.,  361,  328. 
LUBS,  H.  A.,  84,  81. 
LtJDEKiNG,  C.,  319,  362,   292,   294, 

308,  332,  348. 
LUNDEN,  H.,  83,  67. 


LUNDSGAARD,  C.,  218,  211. 

LUTHER,  R.,  271,  266. 

MAAS,  O.,  368,  356. 
MADSEN,  T.,  83,  422,  82,  417,  419. 
MALFATTI,  H.,  163,  143. 
MANABE,  K.,  194,  218,  168,  204. 
MANN,  G.,  32,  135,  163,  318,  21,  109, 

130,  147,  307. 
MARCUS,  E.,  54,  40. 
MARSHALL,  J.  A.,  218,  217. 
MARTIN,  C.  J.,  83,  134,  317,  352,  422, 

70,  123,  129,  305,  307,  309,  340, 

349,  419. 

MASSON,  O.,  362,  323. 
MATHEWS,  A.  P.,  83,  163,  82,  146, 

147. 

MATHEWSON,  W.  E.,  357,  366. 
MATULA,  J.,  31,  54,  194,  217,  218,  23, 

51,  168,  204. 

MAXIMOVICH,  S.,  163,  368,  152,  355. 
MAYER,  K.,  422,  403. 
MAYER,  L.,  218,  213. 
MEDIGRECEANU,  F.,  387,  385. 
MEISSNER,  C.,  422,  389. 
MELLANBY,  J.,  83,  106,  135,  75,  100, 

112. 

MELLOR,  J.  W.,  422,  397. 
MENDEL,  L.  B.,  163,  158,  357. 
MEYER,  K.  F.,  65,  65. 
MEYERS,  C.  N.,  456. 
MICHAELIS,  L.,  83, 105, 164,  218,  387, 

388,  76,  91,  142,  148,  195,  210,  211, 

376,  377,  378,  379,  381,  456. 
MICHAILOV,  W.,  318,  307. 
MIESCHER,  F.,  105,  163,  86,  87,  143, 

146. 

MIGAY,  T.  J.,  422,  418. 
MILROY,  T.  H.,  464,  431. 
MITTELBACH,  F.,  368,  356. 
MIYAKE,  K.,  271,  319,  271,  283,  287. 
MODELSKI,  J.  VON,  33,  20. 
MOLL,  L.,  318,  310. 
MOORE,  A.  R.,  318,  299. 
MOORE,  B.  S.,  135, 194,  362,  127, 169, 

337,  339. 

MORACZEWSKI,  W.,  464,  430. 
MORAWTTZ,  P.,  422,  416,  419. 
MORGENROTH,  J.,  422,  418. 
MORNER,  C.  T.,  64,  368,  49,  355,  356. 
MORNER,  K.  A.  H.,  163,  318, 158, 311. 
MOSTYUSKY,  H.,  218,  210. 
MROCZKOVSKY,  I.,  219,  213. 


474 


INDEX  OF  AUTHORS 


MULLER,  F.,  64,  368,  50,  356. 
MYLIUS,  F.,  163,  143. 

NAGELI,  O.,  194,  193. 

NASSE,  O.,  136,  110. 

NENCKI,  M.,  422,  418. 

NERNST,  W.,  163,  219,  318,  352,  368, 

160,  195,  196,  277,  330,  340,  344, 

347,  464. 

NEUMANN,  J.,  163,  142,  430. 
NEUMEISTER,  R.,  422,  389. 
NIEMANN,  A.,  421,  418. 
NOORDEN,  C.  VON,  368,  358. 
NOTES,  A.  A.,  318,  276. 
NURENBERG,  A.,  163,  157. 
NUTTALL,  G.  H.  F.,  318,  314. 
NYMAN,  M.,  83,  82. 

OBER,  J.  E.,  136,  117,  121. 
OBERMEYER,  F.,  33,  19. 
OKER-BLOM,  M.,  352,  338. 
OKUNOV,  W.  H.,  454,  438. 
OPPENHEIMER,  C.,  163,  388,  422,  454 

156,  369,  434. 
ORYNG,  T.,  194,  169. 
OSBORNE,  T.  B.,  33,  64,  55,  83,  105, 
163,  368,  455,  16,  19,  21,  24,  26,  44, 
46,  48,  49,  71,  101,  102,  103,  133, 
142,  148,  355,  356,  357,  436. 
OSBORNE,  W.  A.,  83,  105,  163,  194, 
250,  271,  318,  27,  71,  88,  90,  148 
169,  193,  234,  253,  282,  284,  464. 
OSTWALD,  WILH.,  136,  271,  352,  422, 

79,  266,  343,  393. 

OSTWALD,  Wo.,  163,  318,  352,  422, 
160,  285,  291,  293,  298,  299,  326 
327,  330,  339,  416. 
OSWALD,  A.,  163,  157,  158. 
OTA,  K.,  135,  125. 
OTTO,  E.,  32,  11. 

PAAL,  C.,  318,  296. 

PALME,  H.,  318,  283. 

PARASTSCHUK,  S.  W.,  422,  418. 

PARKER,  W.  H.,  352,  337. 

PATTEN,  A.  J.,  32,  21. 

PAUL,  T.,  83,  82. 

PAUL,  W.,  317,  310. 

PAULI,  W.,  33, 135, 163, 194,  219,  318, 
368,  23,  107,  108,  111,  112,  119, 
120,  121,  122,  130,  131,  133/134, 
148,  169,  189,  191,  203,  211,  275, 
292,  293,  294,  300,  301,  305,  307, 
309,  310,  339,  358. 


PAVLOV,  I.  P.,  422,  371,  375,  418. 
PECHSTEIN,  H.,  218,  210. 
PELET-JOLIVET,  L.,  147. 
PEMSEL,  W.,  84,  219,  70,  214. 
PFEIFFER,  E.,  422,  415. 
PFEIFFER,  J.  A.  F.,  66,  58,  60. 
PFEIFFER,  P.,  33,  20. 
PICKERING,  S.  U.,  135,  163,  126,  153. 
PICTON,  H.,  135,  112,  113,  114,  115, 

116,  117. 

PINCUSSOHN,  L.,  387,  385. 
PINKUS,  S.  N.,  54,  162,  318,  44,  158, 

311. 

PLANCK,  M.,  362,  330. 
PLATNER,  A.  E.,  83,  67. 
PLIMMER,  R.  H.  A.,  33,  55,  163,  368, 

388,  6,  29,  44,  148,  358,  375. 
PLUCKER,  P.,  352,  349. 
PODOLINSKI,  H.,  422,  415. 
POHL,  J.,  135,  163,  109,  143. 
POLLACK,  L.,  454,  449. 

POSTERNAK,  S.,  135,   120. 

POTILITZIN,  A.,  135,  126. 
POTTEVIN,  H.,  423,  454,  393,  425. 
POYNTING,  J.  H.,  136,  126. 
PRATT,  T.  M.,  65,  105,  56,  97. 
PREYER,  W.  T.,  55,  318,  43,  44,  311. 
PRICE,  T.  M.,  423,  415. 
PROCTER,  H.  R.,  164,  318,  319,  146 

293,  295,  296,  297,  298. 
PROST,  E.,  136,  112,  113,  116. 
PROUST,  M.,  33,  4. 
PUGLIESE,  454,  449. 

QUAGLIARIELLO,  G.,  83,  217,  319,  78, 

204,  309. 

QUINAN,  C.,  65,  40. 
QUINCKE,  H.,  319,  352,  292,  345,  347. 

RABELLO-ALVES,  S.,  161,  350,    142, 

349. 

RAKOCZY,  A.,  423,  454,  418,  438. 
RAMSDEN,  W.,  352,  353. 
RANKINE,  A.  O.,  363,  329. 
RAUDNITZ,  R.  W.,  423,  416. 
REFORMATZKY,  S.,  353,  323. 
REGECZY,  E.  N.,  363,  338. 
REICHEL,  H.,  423,  417. 
REICHERT,  E.  T.,  56,  319,  44,  311, 

313,  314. 

REID,  W.,  164,  353,  160,  332,  337. 
REIGER,  R.,  353,  329. 
REISS,  E.,  66,  368,  60,  359,  360,  365. 


INDEX  OF  AUTHORS 


475 


RENNER,  A.,  362,  328. 

RHORER,  L.  VON,  83,  219,  74,  206. 

RICHARDS,  T.  W.,  194,  193,  308. 

RIGHETTI,  H.,  66,  65. 

RINGER,  S.,  423,  418. 

RINGER,  W.  E.,  194,  219,  168,  204. 

RlTTHAUSEN,  H.,  55,  48. 

ROAF,  H.  E.,  194,  352,  353,  169,  337, 
339. 

ROBERTS,  F.,  161,  160. 

ROBERTSON,  T.  B.,  33,  55,  66,  83, 105, 
106,  136,  162,  164,  194,  219,  250, 
271,  319,  353,  368,  388,  421,  423, 
453,  454,  20,  26,  27,  28,  36,  39,  44, 
46,  49,  51,  56,  60,  65,  67,  69,  70,  73, 
74,  82,  88,  89,  90,  92,  93,  96,  99, 
100,  103,  127,  146,  147,  149,  152, 
154,  155,  160,  170,  171,  177,  178, 
179,  180,  183,  193,  195,  197,  208, 
209,  210,  214,  215,  216,  221,  222, 
223,  224,  225,  228,  234,  237,  238, 
242,  245,  249,  253,  257,  271,  278, 
280,  283,  284,  286,  287,  303,  307, 
333,  336,  339,  340,  341,  346,  347, 
359,  360,  361,  362,  365,  373,  390, 
393,  404,  411,  412,  415,  425,  427, 
428,  432,  434,  436,  437,  442,  445, 
448,  456,  464,  466. 

RODGER,  J.  W.,  271,  266. 

ROGOZINSKI,  F.,  164,  423,  156,  398. 

ROHLOFF,  C.,  353,  329. 

ROHMANN,  F.,  164,  194,  140,  170. 

ROHONYI,  H.,  33,  194,  219,  319,  454, 
26,  168,  204,  304,  435. 

RONA,  P.,  105,  135,  164,  318,  387,  91, 
142,  148,  293,  300,  301. 

ROOSE,  E.,  164,  158. 

ROOZEBOOM,  B.,  123,  124. 

ROSE,  F.,  136,  122. 

ROSENBERG,  H.,  317,  367,  313,  358. 

ROSENBLOOM,  J.,  164,  146. 

ROSENTHALER,  L.,  83,  80. 

ROTH,  W.  A.,  271,  262. 
ROTHMUND,  V.,  136,  130. 
ROWE,  A.  H.,  66,  65. 
RUSSELL,  W.  S.,  136,  126. 
RYDER,  C.  T.,  317,  453,  308,  451. 

SABANEJEV,  A.,  353,  331. 

SACHS,  H.,  454,  449. 

SACKUR,  O.,  54,  105,  194,  218,  251, 

353,  40,  88,  89,  204,  207,  223,  320, 

321,  322,  323,  324. 


SAINSBURY,  H.,  423,  418. 

SALASKIN,  S.,  454. 

SALKOWSKI,  E.,  106,  423,  454,  90, 
389,  431,  432. 

SALM,  E.,  33,  84,  106,  219,  21,  81,  91, 
99,  103,  201,  212. 

SAMEC,  M.,  135,  194,  368,  130,  189, 
191,  358. 

SAMOJLOFF,  A.,  423,  403. 

SAMUELY,  F.,  106,  387,  87. 

SANSUM,  W.  D.,  317,  316. 

SAUNDERS,  E.  R.,  421,  411. 

SAVITSCH,  W.  W.,  422,  423,  418. 

SAVJALOV,  W.,  423,  454,  418,  438. 

SCALINCI,  N.,  317,  299. 

SCHAFER,  E.  A.,  136,  112. 

SCHAFER,  F.,  423,  415. 

SCHEERMESSER,  W.,  368,  356. 

SCHIERBECK,  N.  P.,  423,  415. 

SCHIFF,  H.,  33,  9,  18. 

SCHITTENHELM,  A.,  161,  387,  154. 

SCHLOSSMANN,  A.,  106,  97. 

SCHMIDLIN,  J.,  32,  23. 

SCHMIDT,  A.,  56,  41. 

SCHMIDT,  C.,  423,  410. 

SCHMIDT,  C.  L.  A.,  33,  66,  84,  164, 
219,  319,  353,  368,  423,  454,  30,  65, 
78,  79,  149,  150,  154,  155,  156,  195, 
204,  310,  341,  359,  360,  361,  365, 
411,  412,  442,  456,  457,  463,  466. 

SCHMIDT,  E.  S.,  66,  319,  65,  310. 

SCHMIDT-NIELSEN,  S  and  S.,  423,  418. 

SCHORER,  G.,  66,  368,  65,  362. 

SCHORR,  C.,  363,  327. 

SCHREINEMAKERS,  F.  A.  H.,  136,  124. 

SCHROEDER,  P.  VON,  319,  353,  299, 

323,  326,  327. 
SCHULTZE,   H.,   136,    112,   114,   115, 

116,  132. 
SCHULTZE,  F.  N.,  55,  162,  319,  353, 

51,  311,  316,  350. 
SCHUMM,  O.,  319,  368,  313,  358. 

SCHUMOV-SlMANOVSKY,     E.     O.,     423, 

418. 

SCHUTZ,  E.,  423,  402,  403,  404,  409, 
419,  442. 

SCHUTZENBERGER,  P.,  33,  9. 

SCHWARTZ,  O.,  464,  434,  449. 
SCIPIADES,  E.,  218,  211. 
SCOTT,  L.,  164,  157. 
SEBELJEN,  J.,  368,  355. 
SEEMAN,  T.,  65,  50. 
SERTOLI,  E.,  219,  213,  214. 


476 


INDEX  OF  AUTHORS 


SHARP,  L.  T.,  456. 

SHINJO,  S.,  353,  329. 

SHORTER,  S.  A.,  353,  345,  346,  348. 

SIEBER,  N.,  422,  418. 

SIEGFRIED,  M.,  164,  368,   144,   145, 

356,  358. 
SJOQUIST,  J.,  33,  84,  194,  423,  26,  74, 

170,  403. 

SKRAUP,  H.,  164,  156. 
SLAVU,  161,  154. 
SOLDNER,  F.,  56,  106,  219,  40,  88,  89, 

90,  204,  207. 
SORENSEN,  S.  P.  L.,  33,  84,  219,  319, 

19,  22,  81,  195,  309,  456. 
SORET,  J.  L.,  368,  358,  359. 
SPIRO,  K.,  84,  136,  219,  423,  70,  108, 

123,  214,  417. 
SPRIGGS,  E.  I.,  353,  328. 
STARKE,  J.,  319,  307. 
STARKE,  K.  V.,  368,  355. 
STARLING,  E.  H.,  83,  353,  80,  337. 
STESSANO,  A.,  453,  449. 
STEUDEL,  H.,  55,  423,  50,  389. 
STIEGLITZ,  J.,  388,  423,  384,  393,  395, 

396,  411,  427. 
STILES,  P.  G.,  84,  82. 
STIRLING,  W.,  194,  177,  190. 
STOKES,  G.  C.,  368,  358. 
STRAUSS,  E.,  164,  194,  368,  158,  189, 

191,  358. 

STRECKER,  A.,  84,  67. 
STRONG,  W.  W.,  135,  126. 
SUGIURA,  K.,  32,  163,  22,  142. 
SUIDA,  W.,  164,  148. 
SUTHERLAND,  W.,  251,  319,  353,  230, 

307,  309,  326,  327. 
SZILY,  A.  VON,  219,  211,  212. 

TAMMAN,  G.,  353,  331. 

TANGL,  F.,  319,  351,  455,  308,  332, 
451. 

TAYLOR,  A.  E.,  65, 106,  251,  363,  388, 
423,  424,  465,  40,  54,  87,  241,  341, 
369,  376,  380,  389,  390,  393,  398, 
403,  404,  410,  411,  412,  414,  418, 
425,  427,  428,  439,  447,  448. 

THOMPSON,  W.  B.,  66,  65. 

THOMSON,  J.  J.,  319,  363,  290,  346, 
347. 

THORPE,  T.  E.,  271,  266. 

TIMPE,  H.,  106,  89. 

TITOFF,  A.,  424,  392. 

TJIMSTRA,  S.  B.,  363,  326, 


TOLLOCZKO,  S.,  317,  276. 
TOLMAN,  R.,  136,  125. 
TOOTH,  H.  H.,  421,  415. 
TRANTER,  C.  L.,  66,  65. 
TRUNKEL,  H.,  106,  93. 
TURBUBA,  D.,  424,  392. 

UGGLAS,  B.  AF,  164,  149,  150. 
UHL,  R.,  164,  159. 
UHLER,  H.  S.,  135,  125,  126. 
UMBACH,  T.,  162,  156. 
UMBER,  F.,  455,  438. 
UNDERBILL,  F.  P.,  33,  31. 

VAN  BEMMELEN,  J.  M.,  316,  303. 

VAN  SLYKE,  D.  D.,  31,  33,  55,  106, 
319,  16,  19,  20,  46,  95,  96,  283. 

VAN  SLYKE,  L.  L.,  64,  65,  66,  84, 105, 
106, 219,  261,  319, 424,  37, 40, 46, 56, 
69,  89,  90,  91,  92,  93,  95,  96,  97,  98, 
101,  204,  207,  235,  249,  283,  418. 

VAUBEL,  W.,  162,  157. 

VELEY,  V.  H.,  319,  277. 

VERNON,  H.  M.,  33,  84,  388,  424,  466, 
23,  80,  369,  399,  414,  449. 

VIRCHOW,  R.,  136,  108. 

VOIGTLANDER,  F.,  364,  322,  330. 

VOITINOVICI,  A.,  161,  154. 

WAGNER,  R.,  135,  219,  122,  211. 

WALBUM,  419. 

WALDEN,  P.,  136,  271,  354,  266,  267, 

323. 

WALTER,  A.  A.,  424,  403. 
WALTERS,  E.  H.,  424,  456,  391,  405, 

443. 

WASHBURN,  E.  W.,  460. 
WASILIEV,  N.  P.,  424,  415. 
WEBER,  C.  O.,  354,  327,  342. 
WEBSTER,  A.,  194,  169. 
WEBSTER,  H.  R.,  218,  214. 
WECHSLER,  E.,  164,  158. 
WEINLARD,  E.,  455,  449. 
WEISS,  F.,  32, 106, 163,  424,  8,  22,  86, 

158,  415. 

WELLS,  C.  E.,  66,  65. 
WELLS,  H.  G.,  465,  436. 
WHETHAM,  W.  C.  D.,  134,  136,  364, 

114,  116,  118,323,  464. 
WHITNEY,  W.  R.,  136,  318,  117,  121, 

276. 

WICHMANN,  A.,  319,  311,  315,  316. 
WIEDEMANN,  E.,  319,  292,  294,  308. 


INDEX  OF  AUTHORS 


477 


WIGLOW,  H.,  362,  332. 
WILHEIM,  R.,  32,  19. 
WILSON,  G.  H.,  162,  155. 
WILSON,  J.  A.,  319,  293. 

WlNKELBLECH,     K.,     33,      194,     28, 

191. 

WINTER,  O.  B.,  106,  93. 
WITTICH,  VON,  354,  338. 
WOOD,  T.  B.,  354,  328,  329. 
WOOLSEY,  J.  H.,  66,  65. 
WROBLEWSKI,  A.,  424,  415. 
WYRONBOFF,  M.  G.,  136,  126. 


ZAHN,  F.  W.,  354,  346. 

ZANETTI,  C.  U.,  65,  50. 

ZELINSKY,  N.,  33,  5,  6. 

ZEYNEK,  R.,  319,  311. 

ZIEGLER,  J.,  360,  322. 

ZINNSER,  H.,  164,  154. 

ZINOFFSKI,  O.,  164,  160. 

ZLOBICKI,  L.,  354,  345. 

ZSIGMONDY,  R.,  319,  363,  364,  311, 

316,  349,  350. 
ZUATTRO,  G.  DI,  162,  148. 
ZUNTZ,  N.,  219,  214. 


INDEX  OF  SUBJECTS 


Absorption  of  light,  358. 
Absorption-spectrum,    30,    161,    190, 

358. 

Acidosis,  212,  215. 
Acids,  Compounds  of  proteins  with, 

232 

Adsorption,  118, 147, 160. 
Agglutination,  309. 
Aggregation,  State  of,  275,  366. 
Alcohol,  Coagulation  by,  252. 
Alcohol,  Influence  on  conductivity, 

257. 
Alcohol,   Influence   on   structure   of 

jellies,  301. 

Alcohol-soluble  Proteins,  48. 
Alcohol-water  mixtures,  254. 
Alkaloidal  reagents,  145. 
Alkaloids,  145. 
Amandin,  357. 
Amino-acid  chains,  375. 
Amino-acid  content   and  properties 

of  proteins,  8. 
Amino-acids,  4,  6,  9,  142. 
Ammonium  Caseinate,  222,  223. 
Amphoteric  Acids,  20,  67. 
Amphoteric  character  of  the  proteins, 

81. 

Amygdalin,  393. 
Anaphylaxis,  154,  316. 
Anhydrides, .  10,   29,   130,  268,  309, 

347. 

Antibodies,  154,  156,  436. 
Antigenic  properties,  154,  436. 
Antipepsin,  444,  449. 
Antitrypsin,  444,  449. 
Aromatic     amino-acids,     protective 

action  of,  359. 
Autohydrolysis,  389,  391,  400,  405, 

444. 
Avogadro's  law,  161,  307,  340. 

Bacteria,  82. 
Barium  caseinate,  224. 
Barium  serum  globulinate,  228. 
Benzoyl  derivatives,  156. 
Bicarbonates,  214. 


Bimolecular  reaction  formula,  32. 
Biological  applications,  192,  211,  452. 
Biuret  reaction,  14,  18,  142,  431. 
Blood  serum  analysis,  60,  331. 
Boiling  point,  elevation  of,  331. 

Calcium  caseinate,  224,  230,  261. 
Calcium  salts  in  coagulation  of  milk, 

417. 

Calcium  serum  globulinate,  227. 
Cane  sugar,  79. 

Capillary  forces,  286,  303,  342. 
Carbamino  acids,  144. 
Carbonic  acid,  144. 
Carbon  monoxide,  159. 
Casern,  22,  37,  88,  104,  127,  140,  171, 

198,  204,  243,  271,  343,  346,  349, 

356,  361,  365,  390,  399,  402,  413, 

416,  429,  460. 

Casemates,  27,  232,  237,  320,  333. 
Casemates,  Coagulation  of,  252. 
Caseinates,  Hydrolysis  of,  405,  415. 
Catalysis,  79,  391. 
Catalysors,  389,  426. 
Cerebrospinal  fluid,  60,  350. 
Characterization  of  Proteins,  16. 
Chemical  dynamics,  369. 
Chemical  individuals,  Proteins  as,  34. 
Chemical  statics,  1. 
Chitin,  339. 

Classification,  biological,  311. 
Clupein,  53,  87. 
Coagulating  ferments,  416. 
Coagulating  power  of  salts,  109. 
Coagulation,  68,  108,  119,  252,  299, 

304,  342. 
Coagulation,  Chemical  mechanics  of, 

270. 

Coagulation  of  jellies,  303. 
Coagulation-temperature,  111. 
Coaguloses,  437. 
Cobalt  chloride,  128. 
Cohesiveness,  314,  328. 
Combining  capacity,  73,  93,  195,  204, 

248. 
Combining  weight,  21,  56,  180. 


479 


480 


INDEX  OF  SUBJECTS 


Compounds  of  the  proteins,  67,  85, 

107,  137. 

Compound  proteins,  148. 
Compressibility,  329. 
Concentration-cells,  76,  138,  167. 
Conductivity,  74,  92,  95,  171,  220, 

383,  402,  449,  460. 
Constitution  of  the  proteins,  3. 
Copper  albuminate,  121. 
Copper  sulphate,  124,  133. 
Cryoscopic  method,  74. 
Crystal  habit,  314. 
Crystallization  of  proteins,  43,  44,  47, 

311. 

Crystallin,  355,  356. 
Crystalline  lens,  299. 
Cupric  chloride,  128. 
Cupric  hydrate,  142. 

Deaminized  gelatin,  23,  51,  169,  203. 
Decomposition  of  the  Proteins,  4. 
Degree  of  dissociation,  222,  265,  271, 

322. 

Dehydration,  126,  289,  296,  445. 
Denaturation,  309. 
Denatured  protein,  70. 
Density,  349. 
Deuteroalbumose,  156. 
Diacid  bases,  233. 
Dialysis,  57,  160,  169,  193,  249,  311, 

449. 
Diamino-acids,  5,   17,  25,   144,  176, 

236. 

Diastase,  393. 

Dicarboxylic  acids,  25,  176,  236. 
Diffusion,  277,  304,  322,  330. 
Digestibility,  18. 
Diketopiperazines,  15,  29,  120. 
Dilution,  201,  206,  220. 
Dilution  law,  221,  254,  263. 
Dissociation-constant,  214,  220,  231, 

263. 

Dissociation,  Degree  of,  69. 
Dissociation  of  protein  salts,  167. 
Double  electrical  layer,  114. 
Dyes,  145. 

Edestin,  21,  24,  71,  102,  104,  133,  357, 

365. 
Egg-albumin,  21,  44,  74,  77,  110,  113, 

119,  122,  124,  133,  138,  168,  210, 

305,  311,  328,  331,  337,  349,  350, 

355,  399. 


Egg-globulin,  350. 

Elasticity,  329,  348. 

Electrical  condition,  Influence  of, 
upon  precipitation  and  coagula- 
tion, 112. 

Electrochemical  equivalent,  178,  181, 
182,  268. 

Electrochemical  measurements,  456. 

Electrochemistry,  165. 

Electrolysis,  113,  119,  151,  176. 

Electrostatic  tension,  192,  195,  297. 

Emulsin,  393. 

Energy  storage  in  living  organisms, 
452. 

Enzymatic  synthesis,  439,  448. 

Enzymes,  80. 

Equilibrium,  94,  117,  123,  340,  393, 
396,  425,  439,  447,  451. 

Equivalent  conductivity,  26,  118, 
174,  248. 

Erepsin,  384. 

Excelsin,  357. 

Fats,  enzymatic  synthesis  of,  425. 

Fatty  acids,  148. 

Ferments,    compounds    of    proteins 

with,  156. 
Ferment-substrate    compounds,   380, 

445. 

Ferric  alum,  97. 
Fibrin,  42,  101,  104,  316,  399,  400, 

416. 

Fibrinogen,  356,  416. 
Formic  acid,  127. 
Formol  titration,  19,  437. 
Free  radicals,  192. 
Freezing  point,  Depression  of,  28,  75, 

236,  331. 

Gas  chain,  77,  456. 
Gastric  Juice,  212. 
Gelatin,  51,  110,  203,  292,  301,  322, 

323,  326,  329,  332,  346,  402. 
Gelatinization,  299. 
Gladstone's  law,  362. 
Gliadin,  24,  48,  328,  357,  365. 
Globin,  51,  356,  365. 
Globin  caseinate,  149. 
Glutin,  328. 
Glycerol-water    mixtures,    Viscosity 

of,  322. 

Glycogen,  393. 
Gold  number,  349. 


INDEX  OF  SUBJECTS 


481 


Gorgonin,  158. 
Grotthus  chain,  326. 
Guanidin,  158. 

Haematin,  52,  349,  358. 

Haemoglobin,  14,  43,  159,  305,  311, 
337,  349,  356,  358. 

Haemoglobin  caseinate,  150. 

Halogens,  12: 

Halogen  substitution  compounds, 
157. 

Heat  coagulation,  70,  305. 

Heat,  Liberation  of,  in  protein  hy- 
drolysis, 400. 

Heat,  Liberation  of,  in  swelling,  292. 

Heavy  metals,  121,  137,  159. 

Hemi-albumose,  110. 

Henri's  equation,  380,  406. 

Heterogeneous  systems,  291. 

Homogeneity  of  the  protein  group,  3. 

Honeycomb  structure  of  jellies,  302. 

Hooke's  law,  298,  329. 

Hordein,  357. 

Hybridization,  Influence  of  on  haemo- 
globin crystals,  313. 

Hydantoins,  30,  190. 

Hydration,  108,  119,  125,  129,  411. 

Hydrogen  electrode,  197,  204,  456, 
465. 

Hydrolysis,  4,  14,  23,  57,  280,  371, 
389,  460,  466. 

Hydrolytic  dissociation,  54,  132,  206, 
321. 

Hydrostatic  pressure,  310. 

Hysteresis,  341,  347. 

Imino  groups,  16. 

Immunization,  65,  436. 

Indicators,  56. 

Indicator  method,  81. 

Individuality  of  the  tissues  and  tis- 
sue fluids,  154. 

Infection,  Globulins  in,  65. 

Instability  of  the  proteins,  86. 

Intermediate  compounds  in  catal- 
ysis, 393,  398,  445. 

Internal  salts,  28,  131,  270,  309. 

Iodized  proteins,  156,  157. 

lonization,  Mechanism  of,  325. 

Isoelectric  condition,  209. 

Isohydric  solutions,  203. 

Isolation  of  proteins,  34. 


Isomaltose,  Enzymatic  synthesis  of, 

425. 
Isomorphism,  312. 

Jahn's  law,  138. 

Jellies,  Structure  of,  297,  300. 

Juglansin,  357. 

Kephir  lactase,  393. 
Kinetics  of  hydrolysis,  400. 

Labile  hydrogen  atom,  30,  189. 

Lactalbumin,  355. 

Lactose,  393. 

Leather,  Tanning  of,  146. 

Legumin,  357. 

Lipase,  393. 

Lipoids,  148. 

Magnetic  properties,  Masking  of,  142. 

Magnetic  properties  of  proteins,  349. 

Malt  extract,  403. 

Maltose,  393. 

Masking  of  physiological  effects,  82. 

Mass  law,  116,  201,  220,  340. 

Maxwell's  demon,  299. 

Melanin  nitrogen,  17. 

Methyl  acetate,  Saponification  of,  80. 

Methyl  derivatives,  156. 

Migration  velocity,  26,  184,  187,  220, 

231,  235,  263,  267. 
Milk,  59,  65,  390,  400,  416. 
Millon's  reaction,  14,  431. 
Modulus  of  elasticity,  298. 
Molecular  conductivity,  74,  170. 
Molecular  volume,  331,  343,  362. 
Molecular  weight,  15,  21,  139,  296, 

331,  337,  343. 
Monoamino  acids,  5. 
Monomolecular   reaction,    378,    409, 

444. 
Mucoid,  146. 

Nephelometric  method,  58. 

Network  structure  in  protein  solu- 
tions, 297,  300,  325. 

Neutrality  of  the  tissues  and  tissue 
fluids,  211. 

Neutral  salts,  Influence  of  upon  rate 
of  solution,  283. 

Neutral  salts,  Influence  of  upon  swell- 
ing, 299. 

Nitro  substitution  compounds,  157. 


482 


INDEX  OF  SUBJECTS 


Non-dissociable     inorganic     radical, 

167,  208. 

Non-ionic  protein,  133. 
Non-proteins  in  blood  serum,  60. 
Nucleic  acid,  86,  143. 
Nucleo  proteins,  217,  356. 

(Edema,  299. 

Oligodynamic  action,  193. 

Olive  oil,  Emulsions  of,  303. 

Opalescence,  262,  342. 

Optical  properties  of  protein  solu- 
tions, 355. 

Optical  structure,  373. 

Optimum  temperature,  420. 

Osmometer,  Differential,  337. 

Osmotic  pressure,  160,  196,  294,  330, 
336,  340. 

Ovomucoid,  49,  103,  104,  142,  202, 
246,  336,  350,  356,  365. 

Ovomucoid  chloride,  228,  239. 

Ovomucoid  sulphate,  229. 

Ovovitellin,  44,  72,  365. 

Oxidase,  450. 

Oxygen,  Compounds  of  proteins  with, 
159. 

Pancreatic  juice,  212,  371. 

Paracasein,  97,  416. 

Paramoecia,  Toxicity  of  ultra-violet 

light  for,  359. 
Paranuclein,  36,  155,  365,  393,  427, 

429,  440. 

Penetration,  Coefficient  of,  287. 
Penetration  formula,  287. 
Penetration  of  colloidal  particles,  286. 
Pentavalent  nitrogen,  67. 
Pepsin,  328,  375,  393,  400,  410,  429, 

440. 

Peptids,  15. 

Peptones,  13,  15,  110,  168,  356. 
Permeability  of  protein  films,  348. 
Phaseolin,  357. 
Phase  rule,  123. 
Phenylthiocarbamid,  130. 
Phosphoric  acid,  142,  213. 
Phosphorus,  38,  40,  430. 
Phosphotungstic  acid,  71. 
Phylogenetic  relationships,  312. 
Physical  properties,  273,  320. 
Plasteins,  437. 
Poisseuille's  law,  303. 
Polyamino  acids,  12,  13,  189. 


Polymerization,  268,  289,  309,  336, 
347,  389. 

Polypeptids,  13,  127,  371,  385,  397. 

Polypeptid  structure,  Consequences 
of  the,  20. 

Polysaccharides,  Enzymatic  synthe- 
sis of,  425. 

Potassium  caseinate,  171,  177,  223, 
225,  255,  262,  266. 

Potassium  paranucleinate,  225. 

Potassium  serum  globulinate,  227. 

Potentiometric  method,  76,  93,  99, 
167,  195,  211,  215. 

Precipitating  power,  112. 

Precipitation  and  coagulation,  Chem- 
ical mechanics  of,  125. 

Precipitation,  Indirect  method  of,  71. 

Precipitation  of  proteins,  68,  79,  107. 

Precipitins,  154. 

Principle  of  mobile  equilibrium,  308. 

Proportion  of  inorganic  acid  or  base, 
Influence  of,  on  conductivity,  242. 

Protamin,  22,  53,  86,  346,  393,  403, 
410,  413,  428. 

Protamin  caseinate,  149. 

Protamin  chloride,  229. 

Protamin  edestinate,  155. 

Protamin  sulphate,  229. 

Protein  complexes,  1 52. 

Protein  ions,  173,  176,  184,  321,  323, 
344. 

Purification  of  proteins,  34. 

Quantitative  estimation  of  proteins, 
56. 

Racemized  proteins,  30. 

Radium,  310. 

Reaction  isochore,  160. 

Reaction  velocity,  123,  280,  305,  377, 

381,  401. 

Reciprocal  catalysis,  444. 
Refractive  Index,  92,  359. 
Refractometric  method,  60. 
Rennet,  97,  400,  416,  438. 
Reversion  of  hydrolysis,  425. 
Ricin,  357. 
Rotatory  power,  30,  58,  355,  376,  386. 

Saliva,  217. 

Salmin,  22,  53,  86,  365,  428. 
Salmin  chloride,  229,  241. 
Salmin  sulphate,  229. 


INDEX  OF  SUBJECTS 


483 


Saturation  of  acids  and  bases  by  pro- 
teins, 91,  207,  248. 
Schiitz  rule,  402,  409,  419,  442. 
Scombrin,  87. 

Selective  action  of  living  tissues,  192. 
Serum   albumin,   62,    109,    124,  169, 

203,  311,  328,  345,  349,  355,  365, 

399. 
Serum  globulin,  40,  64,  98,  104,  150, 

187,  349,  356,  365. 
Serum  globulinates,  232. 
Serum  globulin  chloride,  227. 
Serum  proteins,  Neutralizing  power 

of,  215. 

Silicic  acid,  143. 
Silver  nitrate,  124,  138. 
Soaps,  Compounds  of  proteins  with, 

148. 

Sodium  caseinate,  222,  223,  412. 
Sodium  serum  globulinate,  226. 
Solubility,  248,  275. 
Solution,  Passage  into,  275. 
Solution  pressure,  195. 
Solution,  Rate  of,  276. 
Solvate  theory,  130. 
Specific  gravity,  348. 
Specificity,  154,  436. 
Specific  refractivity,  362. 
Spectrophotometric  method,  159. 
Spongework  structure  of  jellies,  300. 
Stoichiometry,  85,  90,  236. 
Strontium  caseinate,  224. 
Strontium  serum  globulinate,  228. 
Structure  of  protein  solutions,  314, 

324. 

Sturin,  22,  53,  87. 
Sulphur,     Compounds     of     proteins 

with,  159. 

Supersaturation,  279,  304,  342. 
Surface  films,  345. 
Surface  tension,  290,  345. 
Suspensions,  341. 
Swelling  deficit,  293. 


Swelling  maximum,  293. 

Swelling  of  jellies,  110,  292,  327,  339, 

416. 

Synaeresis,  316. 
Synthesis,  Enzymatic,  425. 
Synthesis  of  Proteins,  9. 
Syphilis,  350. 

Temperature    coefficient,    160,    284, 

306,  310,  419. 

Tetraethylammonium  iodide,  267. 
Thermodynamics,  337,  450. 
Thermodynamics,  Second  law  of,  299, 

451. 

Thrombin,  316,  416,  450. 
Thymus  histone,  150,  356. 
Thyreoglobulin,  158. 
Thyroid  gland,  158. 
Tissues,  Swelling  of,  299. 
Toxins,  156. 
Triolein,  427. 
Trivalent  carbon,  192. 
Trypsin,  371,  393,  399,  402,  405,  410, 

415,  428. 

Tyndall  effect,  342. 
Types  of  union,  17. 

Ultramicroscope,  316. 
Ultraviolet  light,  310. 
Ultraviolet  light,  Toxic  action  of,  359. 

Valency,  24,  113,  120,  132,  183. 

Vanadium,  142. 

Vegetable  proteins,  46,  101,  311,  357. 

Velocities,  Chemical,  117,  426. 

Viscosity,  210,  266,  320. 

Volume  contraction,  292,  348. 

Yeast,  393. 

Yeast  endotryptase,  387. 

Zein,  48,357. 
Zinc  sulphate,  141. 


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MAY    7   1938 


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